Promoter, promoter control elements, and combinations, and uses thereof

ABSTRACT

The present document is directed to promoter sequences and promoter control elements, polynucleotide constructs comprising the promoters and control elements, and methods of identifying the promoters, control elements, or fragments thereof. The document further relates to the use of such promoters or promoter control elements to modulate transcript levels in plants, and plants containing such promoters or promoter control elements.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2009/038792, filed Mar. 30, 2009, which claims the benefit of U.S.Provisional Application Ser. No. 61/041,018, filed on Mar. 31, 2008. Thedisclosure of the prior applications are incorporated by reference intheir entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING OR TABLE

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying file, named11696-251WO1_sequence_listing was created on Sep. 30, 2010 and is 43.1KB. The file can be accessed using Microsoft Word on a computer thatuses Windows OS.

TECHNICAL FIELD

The present invention relates to promoters and promoter control elementsthat are useful for modulating transcription of a desiredpolynucleotide. Such promoters and promoter control elements can beincluded in polynucleotide constructs, expression cassettes, vectors, orinserted into the chromosome or as an exogenous element, to modulate invivo and in vitro transcription of a polynucleotide. Host cells,including plant cells, and organisms, such as regenerated plantstherefrom, with desired traits or characteristics using polynucleotidescomprising the promoters and promoter control elements described hereinare also a part of the invention.

BACKGROUND

This document relates to promoter sequences and promoter control elementsequences which are useful for the transcription of polynucleotides in ahost cell or transformed host organism.

The introduction of genes into plants has resulted in the development ofplants having new and useful phenotypes such as pathogen resistance,higher levels of healthier types of oils, novel production of healthfulcomponents such as beta-carotene synthesis in rice. An introduced geneis generally a chimeric gene composed of the coding region that confersthe desired trait and regulatory sequences. One regulatory sequence isthe promoter, which is located 5′ to the coding region. This sequence isinvolved in regulating the pattern of expression of a coding region 3′thereof. The promoter sequence binds RNA polymerase complex as well asone or more transcription factors that are involved in producing the RNAtranscript of the coding region.

The promoter region of a gene used in plant transformation is most oftenderived from a different source than is the coding region. It may befrom a different gene of the same species of plant, from a differentspecies of plant, from a plant virus, an algae species, a fungalspecies, or it may be a composite of different natural and/or syntheticsequences. Properties of the promoter sequence generally determine thepattern of expression for the coding region that is operably linked tothe promoter. Promoters with different characteristics of expressionhave been described. The promoter may confer broad expression as in thecase of the widely-used cauliflower mosaic virus (CaMV) 35S promoter.The promoter may confer tissue-specific expression as in the case of theseed-specific phaseolin promoter. The promoter may confer a pattern fordevelopmental changes in expression. The promoter may be induced by anapplied chemical compound, or by an environmental condition applied tothe plant.

The promoter that is used to regulate a particular coding region isdetermined by the desired expression pattern for that coding region,which itself is determined by the desired resulting phenotype in theplant. For example, herbicide resistance is desired throughout the plantso the 35S promoter is appropriate for expression of anherbicide-resistance gene. A seed-specific promoter is appropriate forchanging the oil content of soybean seed. An endosperm-specific promoteris appropriate for changing the starch composition of corn seed. Aroot-specific promoter can be important for improving water or nutrientup-take in a plant. Control of expression of an introduced gene by thepromoter is important because it is sometimes detrimental to haveexpression of an introduced gene in non-target tissues. For example, agene which induces cell death can be expressed in male and/or femalegamete cells in connection with bioconfinement.

One of the primary goals of biotechnology is to obtain organisms, suchas plants, mammals, yeast, and prokaryotes having particular desiredcharacteristics or traits. Examples of these characteristics or traitsabound and may include, for example, in plants, virus resistance, insectresistance, herbicide resistance, enhanced stability or additionalnutritional value. Recent advances in genetic engineering have enabledresearchers in the field to incorporate polynucleotide sequences intohost cells to obtain the desired qualities in the organism of choice.This technology permits one or more polynucleotides from a sourcedifferent than the organism of choice to be transcribed by the organismof choice. If desired, the transcription and/or translation of these newpolynucleotides can be modulated in the organism to exhibit a desiredcharacteristic or trait. Alternatively, new patterns of transcriptionand/or translation of polynucleotides endogenous to the organism can beproduced.

SUMMARY

The present document is directed to isolated polynucleotide sequencesthat comprise promoters and promoter control elements from plants,especially Sorghum bicolor, and other promoters and promoter controlelements functional in plants. It is an object of the present documentto provide isolated polynucleotides that are promoter or promotercontrol sequences. These promoter sequences comprise, for example,

-   -   (1) a polynucleotide having a nucleotide sequence according to        SEQ. ID. NOs. 1-18;    -   (2) a polynucleotide having a nucleotide sequence having at        least 80% sequence identity to a sequence according to SEQ. ID.        NOs. 1-18; and    -   (3) a polynucleotide having a nucleotide sequence which        hybridizes to a sequence according to SEQ. ID. NOs. 1-18 under a        condition establishing a Tm−5° C.

Promoter or promoter control element sequences of the present documentare capable of modulating preferential transcription. In one embodiment,this document features an isolated nucleic acid that includes aregulatory region having 90 percent or greater sequence identity (e.g.,95 percent or greater, or 98 percent or greater) to the nucleotidesequence set forth in any one of SEQ ID NOs. 1-18 or a fragment thereof,wherein the regulatory region directs transcription of an operablylinked heterologous polynucleotide. The nucleic acid can include one ormore motifs selected from the group consisting of an ABRE motif,ABREATRD22 motif, ABRERATCAL motif, ABREZMRAB28 motif,ACGTABREMOTIFA2OSEM motif, ACIIPVPAL2 motif, AGCBOXNPGLB motif, AMYBOX1motif, ARE1 motif, ATHB1ATCONSENSUS motif, ATHB6COREAT motif,AUXRETGA2GMGH3 motif, BOXIIPCCHS motif, CAATBOX1 motif, CACGCAATGMGH3motif, CARGCW8GAT motif, CCA1ATLHCB1 motif, CEREGLUBOX2PSLEGA motif,E2FAT motif, E2FCONSENSUS motif, ERELEE4 motif, GADOWNAT motif,GARE1OSREP1 motif, GAREAT motif, IBOXCORENT motif, INRNTPSADB motif,LRENPCABE motif, MARTBOX motif, MYBGAHV motif, MYBPLANT motif, NRRBNEXTAmotif, P1BS motif, PRECONSCRHSP70A motif, ROOTMOTIFTAPOX1 motif,RYREPEATGMGY2 motif, RYREPEATVFLEB4 motif, SBOXATRBCS motif,SP8BFIBSP8BIB motif, SPHCOREZMC1 motif, TATABOX1 motif, TATABOX2 motif,TATABOX4 motif, TATABOXOSPAL motif, TATCCAYMOTIFOSRAMY3D motif,TE2F2NTPCNA motif, TRANSINITMONOCOTS motif, UP2ATMSD motif, andUPRMOTIFIIAT motif.

This document also features a vector construct that includes a firstnucleic acid that includes a regulatory region having 90 percent orgreater sequence identity (e.g., 95 percent or greater, or 98 percent orgreater) to any one of SEQ ID NOs. 1-18 or a fragment thereof, whereinthe regulatory region directs transcription of an operably linkedheterologous polynucleotide; and a second nucleic acid to betranscribed, wherein the first and second nucleic acids are heterologousto each other and are operably linked. In some embodiments, the firstnucleic acid consists of the nucleic acid set forth in any one of SEQ IDNOs: 1-18. In some embodiments, the second nucleic acid includes anucleic acid sequence that encodes a polypeptide. The second nucleicacid can be operably linked to the first nucleic acid in senseorientation. In some embodiments, the second nucleic acid can betranscribed into an RNA molecule that expresses the polypeptide encodedby the second nucleic acid. The second nucleic acid can be operablylinked to the first nucleic acid in antisense orientation. The secondnucleic acid can be transcribed into an antisense RNA molecule. Thesecond nucleic acid can be transcribed into an interfering RNA againstan endogenous gene.

In another aspect, this document features a transgenic plant or plantcell transformed with an isolated nucleic acid described herein that isoperably linked to a heterologous polynucleotide, or a vector constructdescribed herein. This document also features seeds of such a plant. Insome embodiments, the heterologous nucleic acid encodes a polypeptide ofagronomic interest.

This document also features a method of directing transcription bycombining, in an environment suitable for transcription: a first nucleicacid that includes a regulatory region having 90 percent or greatersequence identity (e.g., 95 percent or greater, or 98 percent orgreater) to any one of SEQ ID NOs. 1-18 or a fragment thereof; and asecond nucleic acid to be transcribed; wherein the first and secondnucleic acids are heterologous to each other and operably linked. Thefirst nucleic acid molecule can consist of a sequence according to anyone of SEQ ID NOs: 1-18. The operably linked first and second nucleicacids can be inserted into a plant cell and the plant cell regeneratedinto a plant.

In yet another aspect, this document features a method of expressing anexogenous coding region in a plant. The method includes transforming aplant cell with a vector described herein; regenerating a stablytransformed plant from the transformed plant cell; and selecting plantscontaining a transformed plant cell, wherein expression of the vectorresults in production of a polypeptide encoded by the second nucleicacid.

This document also features a method of altering the expression of agene in a plant. The method includes transforming a plant cell with anucleic acid described herein that is operably linked to a heterologouspolynucleotide, and regenerating stably transformed plants from thetransformed plant cell. Plants prepared according to such a method alsoare featured, as well as seeds obtained from such plants.

In another aspect, this document features a method of producing atransgenic plant. The method introducing into a plant cell (i) anisolated polynucleotide described herein that is operably linked to aheterologous polynucleotide, or (ii) a vector described herein, andgrowing a plant from the plant cell. The heterologous polynucleotide caninclude a nucleic acid sequence encoding a polypeptide. The heterologouspolynucleotide can be operably linked to the regulatory region in theantisense orientation. The heterologous polynucleotide can betranscribed into an interfering RNA.

In another embodiment, the present promoter control elements are capableof serving as or fulfilling the function, for example, as a corepromoter, a TATA box, a polymerase binding site, an initiator site, atranscription binding site, an enhancer, an inverted repeat, a locuscontrol region, and/or a scaffold/matrix attachment region.

It is yet another object of the present document to provide apolynucleotide that includes at least a first and a second promotercontrol element. The first promoter control element is a promotercontrol element sequence as discussed above, and the second promotercontrol element is heterologous to the first control element; wherein,the first and second control elements are operably linked. Suchpromoters may modulate transcript levels preferentially in a particulartissue or under particular conditions.

In another embodiment, the present isolated polynucleotide comprises apromoter or a promoter control element as described above, wherein thepromoter or promoter control element is operably linked to apolynucleotide to be transcribed.

In another embodiment of the present document, the promoter and promotercontrol elements of the instant document are operably linked to aheterologous polynucleotide that is a regulatory sequence.

It is another object of the present document to provide a host cellcomprising an isolated polynucleotide or vector as described above orfragment thereof. Host cells include, for instance, bacterial, yeast,insect, mammalian, fungus, algae, and plant. The host cell can comprisea promoter or promoter control element exogenous to the genome. Such apromoter can modulate transcription in cis- and in trans-. In yetanother embodiment, the host cell is a plant cell capable ofregenerating into a plant.

It is another object of the present document to provide a method ofmodulating transcription in a sample that contains either a cell-freesystem of transcription or host cell. This method comprises providing apolynucleotide or vector according to the present document as describedabove, and contacting the sample of the polynucleotide or vector withconditions that permit transcription.

In another embodiment of the present method, the polynucleotide orvector preferentially modulates, depending upon the function of theparticular promoter, constitutive transcription, stress inducedtranscription, light induced transcription, dark induced transcription,leaf transcription, root transcription, stem or shoot transcription,silique or fruit transcription, callus transcription, rhizometranscription, stem node transcription, gamete tissue transcription,flower transcription, immature bud or floret and inflorescence specifictranscription, senescing induced transcription, germinationtranscription and/or drought transcription.

Other and further objects of the present document will be made clear orbecome apparent from the following description.

BRIEF DESCRIPTION OF THE TABLES AND FIGURES

The Tables consist of the Expression Reports) for each promoterdescribed herein and provide the nucleotide sequence for each promoterand details for expression driven by each of the nucleic acid promotersequences as observed in transgenic plants. The results are presented assummaries of the spatial expression, which provides information as togross and/or specific expression in various plant organs and tissues.The observed expression pattern is also presented, which gives detailsof expression during different generations or different developmentalstages within a generation. Additional information is provided regardingthe source organism of the promoter, and the vector and marker genesused for the construct. The following symbols are used consistentlythroughout the Tables:

T0: First generation transformant

T1: Second generation transformant

Each row of the table begins with heading of the data to be found in thesection. The following provides a description of the data to be found ineach section:

Heading in Tables Description 1. Promoter tested in: Identifies theorganism in which the promoter-marker vector was tested. 2. Construct:Identifies the promoter by its construct ID 3. Line: Identifies thetransgenic line that contains the promoter construct 4. PromoterCandidate: Provides an internal ID number for the promoter. 5. EventExpressing: Identifies the event numbers that expressed under thepromoter. 6. Spatial expression Identifies the specific parts ofsummary: the plant where various levels of GFP expression are observed.7. Observed expression Provides a general explanation of pattern: whereGFP expression in different generations of plants was observed. 8. Gene:Identifies genomic annotation of the coding sequence that corresponds tothe promoter candidate 9. cDNA I.D.: Internal predicted gene modelcorresponding to the genomic annotation 10. GenBank: pFAM annotationpredicted by GenBank 11. Source Promoter Identifies the plant speciesfrom Organism which the promoter was derived. 12. Vector: Identifies thevector into which a promoter was cloned. 13. Marker Type: Identifies thetype of marker linked to the promoter. The marker is used to determinepatterns of gene expression in plant tissue. 14. Generation screened: T0Identifies the plant generation(s) used Seedling T0 Mature T1 in thescreening process. T0 plants are Seedling primary transformantsregenerated directly from tissue culture while the T1 generation plantsare from the seeds collected from the T0 plants. 16 T0 SeedlingExpression: Identifies plant tissues that were observed for possibleexpression. 17 T0 Mature Plant Identifies plant tissues that wereobserved Expression: for possible expression 18 T1 Mature PlantIdentifies plant tissues that were observed Expression: for possibleexpression 19 Promoter Utility Provides a description of the utility ofthe sequence.

FIG. 1 CRS380_Promoter_EGFP

FIG. 1 is a schematic representation of a vector that is useful toinsert promoters described herein into a plant. The definitions of theabbreviations used in the vector map are as follows: Ori—the origin ofreplication used by an E. coli host; RB—sequence for the right border ofthe T-DNA from pMOG800; SfiI—restriction enzyme cleavage site used forcloning; EGFP—an enhanced version of the green fluorescent protein gene;OCS—the terminator sequence from the octopine synthase gene; p28716(a.k.a 28716 short)—promoter used to drive expression of the PAT (BAR)gene; PAT (BAR)—a marker gene conferring herbicide resistance;LB—sequence for the left border of the T-DNA from pMOG800; Spec—a markergene conferring spectinomycin resistance; TrfA—transcription repressionfactor gene; RK2-OriV—origin of replication for Agrobacterium.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The document disclosed herein provides promoters capable of driving theexpression of an operably linked transgene. The design, construction,and use of these promoters is one object of this document. The promotersequences, SEQ ID NOs: 1-18, are capable of transcribing operably linkednucleic acid molecules in particular plant tissues/organs or duringparticular plant growth stages, and therefore can selectively regulateexpression of transgenes in these tissues/organs or at these times ofplant development.

1. DEFINITIONS

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and refer to both RNA and DNA, including cDNA, genomic DNA,synthetic DNA, and DNA or RNA containing nucleic acid analogs.Polynucleotides can have any three-dimensional structure. A nucleic acidcan be double-stranded or single-stranded, i.e., a sense strand or anantisense strand. Non-limiting examples of polynucleotides includegenes, gene fragments, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers, as well as nucleic acid analogs.

An isolated nucleic acid can be, for example, a naturally-occurring DNAmolecule, provided one of the nucleic acid sequences normally foundimmediately flanking that DNA molecule in a naturally-occurring genomeis removed or absent. Thus, an isolated nucleic acid includes, withoutlimitation, a DNA molecule that exists as a separate molecule,independent of other sequences, e.g., a chemically synthesized nucleicacid, or a cDNA or genomic DNA fragment produced by the polymerase chainreaction (PCR) or restriction endonuclease treatment. An isolatednucleic acid also refers to a DNA molecule that is incorporated into avector, an autonomously replicating plasmid, or a virus, or transformedinto the genome of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

Chimeric: The term “chimeric” is used to describe polynucleotides orgenes, or constructs wherein at least two of the elements of thepolynucleotide or gene or construct, such as the promoter and thepolynucleotide to be transcribed and/or other regulatory sequencesand/or filler sequences and/or complements thereof, are heterologous toeach other.

Broadly Expressing Promoter: Promoters referred to herein as “broadlyexpressing promoters” actively promote transcription under most, but notnecessarily all, environmental conditions and states of development orcell differentiation. Examples of broadly expressing promoters includethe cauliflower mosaic virus (CaMV) 35S transcript initiation region andthe 1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens,and other transcription initiation regions from various plant genes,such as the maize ubiquitin-1 promoter, known to those of skill.

Domain: Domains are fingerprints or signatures that can be used tocharacterize protein families and/or parts of proteins. Suchfingerprints or signatures can comprise conserved (1) primary sequence,(2) secondary structure, and/or (3) three-dimensional conformation. Asimilar analysis can be applied to polynucleotides. Generally, eachdomain has been associated with either a conserved primary sequence or asequence motif. Generally these conserved primary sequence motifs havebeen correlated with specific in vitro and/or in vivo activities. Adomain can be any length, including the entirety of the polynucleotideto be transcribed. Examples of domains include, without limitation, AP2,helicase, homeobox, zinc finger, etc.

Endogenous: The term “endogenous,” within the context of the currentdocument refers to any polynucleotide, polypeptide or protein sequencewhich is a natural part of a cell or organism(s) regenerated from saidcell. In the context of promoter, the term “endogenous coding region” or“endogenous cDNA” refers to the coding region that is naturally operablylinked to the promoter.

Enhancer/Suppressor: An “enhancer” is a DNA regulatory element that canincrease the steady state level of a transcript, usually by increasingthe rate of transcription initiation. Enhancers usually exert theireffect regardless of the distance, upstream or downstream location, ororientation of the enhancer relative to the start site of transcription.In contrast, a “suppressor” is a corresponding DNA regulatory elementthat decreases the steady state level of a transcript, again usually byaffecting the rate of transcription initiation. The essential activityof enhancer and suppressor elements is to bind a protein factor(s). Suchbinding can be assayed, for example, by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in an in vitro transcription extract.

Exogenous: As referred to within, “exogenous” is any polynucleotide,polypeptide or protein sequence, whether chimeric or not, that isintroduced into the genome of a host cell or organism regenerated fromsaid host cell by any means other than by a sexual cross. Examples ofmeans by which this can be accomplished are described below, and includeAgrobacterium-mediated transformation (of dicots—e.g. Salomon et al.(1984) EMBO J. 3:141; Herrera-Estrella et al. (1983) EMBO J. 2:987; ofmonocots, representative papers are those by Escudero et al. (1996)Plant J. 10:355), Ishida et al. (1996) Nature Biotech 14:745, May et al.(1995) Bio/Technology 13:486), biolistic methods (Armaleo et al. (1990)Current Genetics 17:97), electroporation, in planta techniques, and thelike. Such a plant containing the exogenous nucleic acid is referred tohere as a T₀ for the primary transgenic plant and T₁ for the firstgeneration. The term “exogenous” as used herein is also intended toencompass inserting a naturally found element into a non-naturally foundlocation.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if other filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Homologous: In the current document, a “homologous” polynucleotiderefers to a polynucleotide that shares sequence similarity with thepolynucleotide of interest. This similarity may be in only a fragment ofthe sequence and often represents a functional domain such as, examplesincluding, without limitation, a DNA binding domain or a domain withtyrosine kinase activity. The functional activities of homologouspolynucleotides are not necessarily the same.

Inducible Promoter: An “inducible promoter” in the context of thecurrent document refers to a promoter, the activity of which isinfluenced by certain conditions, such as light, temperature, chemicalconcentration, protein concentration, conditions in an organism, cell,or organelle, etc. A typical example of an inducible promoter, which canbe utilized with the polynucleotides of the present document, is PARSK1,the promoter from an Arabidopsis gene encoding a serine-threonine kinaseenzyme, and which promoter is induced by dehydration, abscissic acid andsodium chloride (Wang and Goodman (1995) Plant J. 8:37). Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, elevated temperature, thepresence or absence of a nutrient or other chemical compound or thepresence of light.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome, including a gene or coding region from a different plantspecies or from a non-plant organism.

Modulate Transcription Level: As used herein, the phrase “modulatetranscription” describes the biological activity of a promoter sequenceor promoter control element. Such modulation includes, withoutlimitation, up- and down-regulation of initiation of transcription, rateof transcription, and/or transcription levels.

Operable Linkage: An “operable linkage” is a linkage in which a promotersequence or promoter control element is connected to a polynucleotidesequence (or sequences) in such a way as to place transcription of thepolynucleotide sequence under the influence or control of the promoteror promoter control element. Two DNA sequences (such as a polynucleotideto be transcribed and a promoter sequence linked to the 5′ end of thepolynucleotide to be transcribed) are said to be operably linked ifinduction of promoter function results in the transcription of mRNAencoding the polynucleotide and if the nature of the linkage between thetwo DNA sequences does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the promotersequence to direct the expression of the protein, antisense RNA, RNAi orribozyme, or (3) interfere with the ability of the DNA template to betranscribed. Thus, a promoter sequence would be operably linked to apolynucleotide sequence if the promoter was capable of effectingtranscription of that polynucleotide sequence.

Percentage of sequence identity As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence and a subject sequence. A subject sequence typically hasa length that is from about 80 percent to 250 percent of the length ofthe query sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105,110, 115, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, or 250 percent of the length of the query sequence. A query nucleicacid or amino acid sequence is aligned to one or more subject nucleicacid or amino acid sequences using the computer program ClustalW(version 1.83, default parameters), which allows alignments of nucleicacid or protein sequences to be carried out across their entire length(global alignment). Chenna et al. (2003) Nucleic Acids Res.31(13):3497-500.

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For an alignment of multiple nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launcherwebsite and at the European Bioinformatics Institute website on theWorld Wide Web.

To determine a percent identity for polypeptide or nucleic acidsequences between a query and a subject sequence, the sequences arealigned using Clustal W and the number of identical matches in thealignment is divided by the query length, and the result is multipliedby 100. The output is the percent identity of the subject sequence withrespect to the query sequence. It is noted that the percent identityvalue can be rounded to the nearest tenth. For example, 78.11, 78.12,78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17,78.18, and 78.19 are rounded up to 78.2.

Plant Promoter: A “plant promoter” is a promoter capable of initiatingtranscription in plant cells and can modulate transcription of apolynucleotide. Such promoters need not be of plant origin. For example,promoters derived from plant viruses, such as the CaMV35S promoter orfrom Agrobacterium tumefaciens such as the T-DNA promoters, can be plantpromoters. A typical example of a plant promoter of plant origin is themaize ubiquitin-1 (ubi-1) promoter known to those of skill in the art.

Plant Tissue: The term “plant tissue” includes differentiated andundifferentiated tissues or plants, including but not limited to roots,stems, shoots, rhizomes, cotyledons, epicotyl, hypocotyl, leaves,pollen, seeds, gall tissue and various forms of cells in culture such assingle cells, protoplast, embryos, and callus tissue. The plant tissuemay be in plants or in organ, tissue or cell culture.

Preferential Transcription: “Preferential transcription” is defined astranscription that occurs in a particular pattern of cell types ordevelopmental times or in response to specific stimuli or combinationthereof. Non-limitive examples of preferential transcription include:high transcript levels of a desired sequence in root tissues; detectabletranscript levels of a desired sequence in certain cell types duringembryogenesis; and low transcript levels of a desired sequence underdrought conditions. Such preferential transcription can be determined bymeasuring initiation, rate, and/or levels of transcription.

Promoter: A “promoter” is a DNA sequence that directs the transcriptionof a polynucleotide. Typically a promoter is located in the 5′ region ofa polynucleotide to be transcribed, proximal to the transcriptionalstart site of such polynucleotide. More typically, promoters are definedas the region upstream of the first exon; more typically, as a regionupstream of the first of multiple transcription start sites; moretypically, as the region downstream of the preceding gene and upstreamof the first of multiple transcription start sites; more typically, theregion downstream of the polyA signal and upstream of the first ofmultiple transcription start sites; even more typically, about 3,000nucleotides upstream of the ATG of the first exon; even more typically,2,000 nucleotides upstream of the first of multiple transcription startsites. The promoters of the document comprise at least a core promoteras defined above. Frequently promoters are capable of directingtranscription of genes located on each of the complementary DNA strandsthat are 3′ to the promoter. Stated differently, many promoters exhibitbidirectionality and can direct transcription of a downstream gene whenpresent in either orientation (i.e. 5′ to 3′ or 3′ to 5′ relative to thecoding region of the gene). Additionally, the promoter may also includeat least one control element such as an upstream element. Such elementsinclude UARs and optionally, other DNA sequences that affecttranscription of a polynucleotide such as a synthetic upstream element.

Promoter Control Element: The term “promoter control element” as usedherein describes elements that influence the activity of the promoter.Promoter control elements include transcriptional regulatory sequencedeterminants such as, but not limited to, enhancers, scaffold/matrixattachment regions, TATA boxes, transcription start locus controlregions, UARs, URRs, other transcription factor binding sites andinverted repeats.

Public sequence: The term “public sequence,” as used in the context ofthe instant application, refers to any sequence that has been depositedin a publicly accessible database prior to the filing date of thepresent application. This term encompasses both amino acid andnucleotide sequences. Such sequences are publicly accessible, forexample, on the BLAST databases on the NCBI FTP web site (accessible viathe internet). The database at the NCBI FTP site utilizes “gi” numbersassigned by NCBI as a unique identifier for each sequence in thedatabases, thereby providing a non-redundant database for sequence fromvarious databases, including GenBank, EMBL, DBBJ (DNA Database of Japan)and PDB (Brookhaven Protein Data Bank).

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns. Regulatory regions can beclassified in two categories, promoters and other regulatory regions.

Regulatory Sequence: The term “regulatory sequence,” as used in thecurrent document, refers to any nucleotide sequence that influencestranscription or translation initiation and rate, or stability and/ormobility of a transcript or polypeptide product. Regulatory sequencesinclude, but are not limited to, promoters, promoter control elements,protein binding sequences, 5′ and 3′ UTRs, transcriptional start sites,termination sequences, polyadenylation sequences, introns, certainsequences within amino acid coding sequences such as secretory signals,protease cleavage sites, etc.

Specific Promoters: In the context of the current document, “specificpromoters” refers to a subset of promoters that have a high preferencefor modulating transcript levels in a specific tissue, or organ or celland/or at a specific time during development of an organism. By “highpreference” is meant at least 3-fold, preferably 5-fold, more preferablyat least 10-fold still more preferably at least 20-fold, 50-fold or100-fold increase in transcript levels under the specific condition overthe transcription under any other reference condition considered.Typical examples of temporal and/or tissue or organ specific promotersof plant origin that can be used with the polynucleotides of the presentdocument, are: PTA29, a promoter which is capable of driving genetranscription specifically in tapetum and only during anther development(Koltonow et al. (1990) Plant Cell 2:1201; RCc2 and RCc3, promoters thatdirect root-specific gene transcription in rice (Xu et al. (1995) PlantMol. Biol. 27:237; TobRB27, a root-specific promoter from tobacco(Yamamoto et al. (1991) Plant Cell 3:371). Examples of tissue-specificpromoters under developmental control include promoters that initiatetranscription only in certain tissues or organs, such as root, ovule,fruit, seeds, or flowers. Other specific promoters include those fromgenes encoding seed storage proteins or the lipid body membrane protein,oleosin. A few root-specific promoters are noted above. See also“Preferential transcription.”

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)−5° C. to T_(m)−10° C. Medium or moderate stringencyconditions are those providing T_(m)−20° C. to T_(m)−29° C. Lowstringency conditions are those providing a condition of T_(m)−40° C. toT_(m)−48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:T _(m)=81.5−16.6(log₁₀ [Na⁺])+0.41(% G+C)−(600/N)  (I)

where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(%formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid(21). The T_(m) of Equation II is affected by the nature of the hybrid:for DNA-RNA hybrids, T_(m) is 10-15° C. higher than calculated; forRNA-RNA hybrids, T_(m) is 20-25° C. higher. Because the T_(m) decreasesabout 1° C. for each 1% decrease in homology when a long probe is used(Frischauf et al. (1983) J. Mol Biol, 170: 827-842), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present document are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m) medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the T₀ plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

TATA to start: “TATA to start” shall mean the distance, in number ofnucleotides, between the primary TATA motif and the start oftranscription.

Transgenic plant: A “transgenic plant” is a plant having one or moreplant cells that contain at least one exogenous polynucleotideintroduced by recombinant nucleic acid methods.

Translational start site: In the context of the present document, a“translational start site” is usually an ATG or AUG in a transcript,often the first ATG or AUG. A single protein encoding transcript,however, may have multiple translational start sites.

Transcription start site: “Transcription start site” is used in thecurrent document to describe the point at which transcription isinitiated. This point is typically located about 25 nucleotidesdownstream from a TFIID binding site, such as a TATA box. Transcriptioncan initiate at one or more sites within the gene, and a singlepolynucleotide to be transcribed may have multiple transcriptional startsites, some of which may be specific for transcription in a particularcell-type or tissue or organ. “+1” is stated relative to thetranscription start site and indicates the first nucleotide in atranscript.

Upstream Activating Region (UAR): An “Upstream Activating Region” or“UAR” is a position or orientation dependent nucleic acid element thatprimarily directs tissue, organ, cell type, or environmental regulationof transcript level, usually by affecting the rate of transcriptioninitiation. Corresponding DNA elements that have a transcriptioninhibitory effect are called herein “Upstream Repressor Regions” or“URR”s. The essential activity of these elements is to bind a proteinfactor. Such binding can be assayed by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in vitro transcription extract.

Untranslated region (UTR): Untranslated region (UTR): A “UTR” is anycontiguous series of nucleotide bases that is transcribed, but is nottranslated. A 5′ UTR lies between the start site of the transcript andthe translation initiation codon (ATG codon) and includes the +1nucleotide of the messenger RNA or cDNA. Alternately, 5′ UTR can besynthetically produced or manipulated DNA elements. A “plant 5′ UTR” canbe a native or non-native 5′ UTR that is functional in plant cells. A 5′UTR can be used as a 5′ regulatory element for modulating expression ofan operably linked transcribable polynucleotide molecule. For example,5′ UTRs derived from heat shock protein genes have been demonstrated toenhance gene expression in plants (see for example, U.S. Pat. No.5,659,122 and U.S. Pat. No. 5,362,865, all of which are incorporatedherein by reference). Examples of 5′ UTRs include those shown in SEQ IDNOs:1-7, 9-12, 16-18. A 3′ UTR lies between the translation terminationcodon and the end of the transcript. UTRs can have particular functionssuch as increasing mRNA message stability or translation attenuation.Examples of 3′ UTRs include, but are not limited to polyadenylationsignals and transcription termination sequences.

2. USE OF THE PROMOTERS

The promoters and promoter control elements of this document are capableof modulating transcription. Such promoters and promoter controlelements can be used in combination with native or heterologous promoterfragments, control elements or other regulatory sequences to modulatetranscription and/or translation.

Specifically, promoters and control elements of the document can be usedto modulate transcription of a desired polynucleotide, which includeswithout limitation:

-   -   a) antisense;    -   b) ribozymes;    -   c) coding sequences; or    -   d) fragments thereof.

The promoter also can modulate transcription in a host genome in cis- orin trans-.

In an organism, such as a plant, the promoters and promoter controlelements of the instant document are useful to produce preferentialtranscription which results in a desired pattern of transcript levels ina particular cells, tissues, or organs, or under particular conditions.

3. IDENTIFYING AND ISOLATING PROMOTER SEQUENCES

The promoters and promoter control elements of the present document arepresented in the Promoter Reports of the Tables and were identified fromSorghum bicolor. Isolation from genomic libraries of polynucleotidescomprising the sequences of the promoters and promoter control elementsof the present document is possible using known techniques. For example,polymerase chain reaction (PCR) can amplify the desired polynucleotidesutilizing primers designed from SEQ ID NOs: 1-18. Polynucleotidelibraries comprising genomic sequences can be constructed according toSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.(1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), for example.

Other procedures for isolating polynucleotides comprising the promotersequences of the document include, without limitation, tail-PCR, and 5′rapid amplification of cDNA ends (RACE). See, for tail-PCR, for example,Liu et al. (1995) Plant J 8(3): 457-463; Liu et al. (1995) Genomics 25:674-681; Liu et al. (1993) Nucl. Acids Res. 21(14): 3333-3334; and Zoeet al. (1999) BioTechniques 27(2): 240-248; for RACE, see, for example,PCR Protocols: A Guide to Methods and Applications, (1990) AcademicPress, Inc.

In addition, the promoters and promoter control elements described inthe Promoter Reports in the Tables (SEQ. ID. Nos. 1-18) can bechemically synthesized according to techniques in common use. See, forexample, Beaucage et al. (1981) Tet. Lett. 22: 1859 and U.S. Pat. No.4,668,777. Such chemical oligonucleotide synthesis can be carried outusing commercially available devices, such as, Biosearch 4600 or 8600DNA synthesizer, by Applied Biosystems, a division of Perkin-ElmerCorp., Foster City, Calif., USA; and Expedite by Perceptive Biosystems,Framingham, Mass., USA.

Included in the present document are promoters exhibiting nucleotidesequence identity to SEQ. ID. Nos. 1-18. In particular, promoters ofthis document can exhibit at least 80% sequence identity (e.g., at least85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity)compared to the nucleotide sequence set forth in any one of SEQ. ID.Nos. 1-18. Sequence identity can be calculated by the algorithms andcomputers programs described above. Furthermore, promoters describedherein also can be a fragment of any one of SEQ ID NO:1-18 as long asthe fragment retains the ability to direct transcription of apolynucleotide. Suitable fragments can be, for example at least 80%(e.g., at least 85, 90, 95, 96, 97, 98, or 99%) of the length of thenucleotide sequence set forth in any one of SEQ ID NOs:1-18. Forexample, a regulatory region can be a fragment of anyone of SEQ IDNOs:1-18 that is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,or 2400 nucleotides in length that retains the ability to directexpression of an operably linked nucleic acid.

A regulatory region can contain conserved regulatory motifs. Such aregulatory region can be have a nucleotide sequence set forth in anyoneof SEQ ID NOs:1-18, or a regulatory region having a nucleotide sequencethat deviates from that set forth in SEQ ID NOs:1-18, while retainingthe ability to direct expression of an operably linked nucleic acid. Forexample, a regulatory region can contain a CAAT box or a TATA box. ACAAT box is a conserved nucleotide sequence involved in initiation oftranscription. A CAAT box functions as a recognition and binding sitefor regulatory proteins called transcription factors. A TATA box isanother conserved nucleotide sequence involved in transcriptioninitiation. A TATA box seems to be important in determining accuratelythe position at which transcription is initiated.

Other conserved regulatory motifs can be identified using methods knownin the art. For example, a regulatory region can be analyzed using thePLACE (PLAnt Cis-acting regulatory DNA Elements) Web Signal Scan programon the world wide web at dna.affrc.go.jp/PLACE/signalscan.html. See,Higo et al., Nucleic Acids Research, 27(1):297-300 (1999); andPrestridge, CABIOS, 7:203-206 (1991). Examples of conserved regulatorymotifs can be found in the PLACE database on the world wide web atdna.affrc.go.jp/PLACE/. See, Higo et al., supra.

A regulatory region having a nucleotide sequence set forth in anyone ofSEQ ID NOs:1-18, or a regulatory region having a nucleotide sequencethat deviates from that set forth in SEQ ID NOs:1-18, while retainingthe ability to direct expression of an operably linked nucleic acid, cancontain one or more conserved regulatory motifs, which can be found inthe PLACE database. For example, a regulatory region can contain an ABREmotif having the consensus sequence ACGTG. See, Simpson et al., Plant J.33: 259-270 (2003); Nakashima et al., Plant Mol Biol. 60:51-68 (2006). Aregulatory region can contain an ABREATRD22 motif having the consensussequence RYACGTGGYR (SEQ ID NO:19). See, Iwasaki et al., Mol Gen Genet247:391-398 (1995); Bray, Trends Plant Sci. 2:48-54 (1997); Busk andPages, Plant Mol Biol 37:425-435 (1998). A regulatory region can containan ABRERATCAL motif having the consensus sequence MACGYGB. See, Kaplanet al., Plant Cell. 18:2733-2748 (2006). A regulatory region can containan ABREZMRAB28 motif having the consensus sequence CCACGTGG. See, Suzukiet al., Plant Physiol. 139: 437-447 (2005); Busk and Pages, Plant MolBiol 37:425-435 (1998); Alonso-Blanco et al., Plant Physiology 139:1304-1312 (2005); Guan et al., Plant J 22: 87-95 (2000); Benedict etal., Plant Cell Environ. 29:1259-1272 (2006); Jaglo-Ottosen et al.,Science 1998 3: 104-106 (1998); and Gilmour et al., Plant J 16: 433-442(1998). A regulatory region can contain an ACGTABREMOTIFA2OSEM motifhaving the consensus sequence ACGTGKC. See, Hattori et al., Plant CellPhysiol 43: 136-140 (2002); and Narusaka et al., Plant J 34: 137-148(2003). A regulatory region can contain an ACIIPVPAL2 motif having theconsensus sequence CCACCAACCCCC (SEQ ID NO:20). See, Patzlaff et al.,Plant Mol Biol. 53:597-608 (2003); Hatton et al., Plant J 7:859-876(1995); and Gomez-Maldonado et al., Plant J. 39:513-526 (2004). Aregulatory region can contain an AGCBOXNPGLB motif having the consensussequence TATTCT. See, Hart et al., Plant Mol Biol 21:121-131 (1993);Fujimoto et al., Plant Cell 12:393-404 (2000); Sato et al., Plant CellPhysiol 37: 249-255 (1996); Ohme-Takagi et al., Plant Cell Physiol 41:1187-1192 (2000); Rushton et al., Plant Cell 14: 749-762 (2002); Cheonget al., Plant Physiol. 132: 1961-1972 (2003); and Zhang et al., Planta.220: 262-270 (2004). A regulatory region can contain an AMYBOX1 motifhaving the consensus sequence TAACARA. See, Huang et al., Plant Mol Biol14:655-668 (1990). A regulatory region can contain an ARE1 motif havingthe consensus sequence RGTGACNNNGC (SEQ ID NO:21). See, Rushmore et al.,J Biol Chem 266:11632-11639 (1991). A regulatory region can contain anATHB1ATCONSENSUS motif having the consensus sequence CAATWATTG. See,Sessa et al., EMBO J. 12:3507-3517 (1993). A regulatory region cancontain an ATHB6COREAT motif having the consensus sequence CAATTATTA.See, Himmelbach et al., EMBO J 21:3029-3038 (2002). A regulatory regioncan contain an AUXRETGA2GMGH3 motif having the consensus sequenceTGACGTAA. See, Liu et al., Plant Cell 6:645-657 (1994); Liu et al.,Plant Physiol 115:397-407 (1997); and Guilfoyle et al., Plant Physiol118: 341-347 (1998). A regulatory region can contain a BOXIIPCCHS motifhaving the consensus sequence ACGTGGC. See, Block et al., Proc Natl AcadSci USA 87:5387-5391 (1990); Terzaghi and Cashmore, Annu Rev PlantPhysiol Plant Mol Biol 46:445-474 (1995); and Nakashima et al., PlantMol Biol. 60: 51-68 (2006). A regulatory region can contain a CAATBOX1motif having the consensus sequence CCAAT. See, Shirsat et al., Mol GenGenet 215:326-331 (1989). A regulatory region can contain aCACGCAATGMGH3 motif having the consensus sequence CACGCAAT. See, Ulmasovet al., Plant Cell 7: 1611-1623 (1995). A regulatory region can containa CARGCW8GAT motif having the consensus sequence CWWWWWWWWG (SEQ IDNO:22). See, Tang and Perry, J Biol Chem. 278:28154-28159 (2003); Folterand Angenent, Trends Plant Sci. 11:224-231 (2006). A regulatory regioncan contain a CCA1ATLHCB1 motif having the consensus sequence AAMAATCT.See, Wang et al., Plant Cell 9:491-507 (1997). A regulatory region cancontain a CEREGLUBOX2PSLEGA motif having the consensus sequenceTGAAAACT. See, Shirsat et al., supra. A regulatory region can contain anE2FAT motif having the consensus sequence TYTCCCGCC. See, Ramirez-Parraet al., Plant J. 33: 801-811 (2003). A regulatory region can contain anE2FCONSENSUS motif having the consensus sequence WTTSSCSS. See,Vandepoele et al., Plant Physiol. 139: 316-328 (2005). A regulatoryregion can contain an ERELEE4 motif having the consensus sequenceAWTTCAAA. See, Itzhaki et al., Proc Natl Acad Sci USA 91:8925-8929(1994); Montgomery et al., Proc Natl Acad Sci USA 90:5939-5943 (1993);Tapia et al., Plant Physiol. 138:2075-2086 (2005); and Rawat et al.,Plant Mol Biol. 57: 629-643 (2005). A regulatory region can contain aGADOWNAT motif having the consensus sequence ACGTGTC. See, Ogawa et al.,Plant Cell 15: 1591-1604 (2003); and Nakashima et al., Plant Mol Biol.60: 51-68 (2006). A regulatory region can contain a GARE1OSREP1 motifhaving the consensus sequence TAACAGA. See, Sutoh and Yamauchi, Plant J.34: 636-645 (2003). A regulatory region can contain a GAREAT motifhaving the consensus sequence TAACAAR. See, Ogawa et al., Plant Cell 15:1591-1604 (2003). A regulatory region can contain an IBOXCORENT motifhaving the consensus sequence GATAAGR. See, Martinez-Hernandez et al.,Plant Physiol. 128:1223-1233 (2002). A regulatory region can contain anINRNTPSADB motif having the consensus sequence YTCANTYY. See, Nakamuraet al., Plant J 29: 1-10 (2002). A regulatory region can contain aLRENPCABE motif having the consensus sequence ACGTGGCA. See, Castresanaet al., EMBO J. 7:1929-1936 (1988). A regulatory region can contain aMARTBOX motif having the consensus sequence TTWTWTTWTT (SEQ ID NO:23).See, Gasser et al., Int Rev Cyto 119:57-96 (1989). A regulatory regioncan contain a MYBGAHV motif having the consensus sequence TAACAAA. See,Gubler et al., Plant Cell 7:1879-1891 (1995); Morita et al., FEBS Lett423:81-85 (1998); Gubler et al., Plant J. 17:1-9 (1999). A regulatoryregion can contain a MYBPLANT motif having the consensus sequenceMACCWAMC. See, Sablowski et al., EMBO J 13:128-137 (1994); Tamagnone etal., Plant Cell 10: 135-154 (1998). A regulatory region can contain aNRRBNEXTA motif having the consensus sequence TAGTGGAT. See, Elliott andShirsat, Plant Mol Biol 37:675-687 (1998). A regulatory region cancontain a P1BS motif having the consensus sequence GNATATNC. See, Rubioet al., Genes Dev. 15: 2122-2133. (2001); Shunmann et al., J Exp Bot.55: 855-865. (2004); and Shunmann et al., Plant Physiol. 136: 4205-4214(2004). A regulatory region can contain a PRECONSCRHSP70A motif havingthe consensus sequence SCGAYNRNNNNNNNNNNNNNNNHD (SEQ ID NO:24). See, vonGromoff et al., Nucleic Acids Res. 34:4767-4779 (2006). A regulatoryregion can contain a ROOTMOTIFTAPOX1 motif having the consensus sequenceATATT. See, Elmayan and Tepfer, Transgenic Res 4:388-396 (1995). Aregulatory region can contain a RyREPEATGMGY2 motif having the consensussequence CATGCAT. See, Lelievre et al., Plant Physiol 98:387-391 (1992).A regulatory region can contain a RyREPEATVFLEB4 motif having theconsensus sequence CATGCATG. See, Curaba et al., Plant Physiol. 136:3660-3669 (2004); and Nag et al., Plant Mol Biol. 59: 821-838 (2005). Aregulatory region can contain a SBOXATRBCS motif having the consensussequence CACCTCCA. See, Acevedo-Hernandez et al., Plant J. 43:506-519(2005). A regulatory region can contain a SP8BFIBSP8BIB motif having theconsensus sequence TACTATT. See, Ishiguro and Nakamura, Plant Mol Biol18:97-108 (1992); Ishiguro and Nakamura, Mol Gen Genet. 244: 563-571(1994). A regulatory region can contain a TATABOX1, TATABOX2, orTATABOX4 motif having the consensus sequence CTATAAATAC (SEQ ID NO:25),TATAAAT, and TATATAA, respectively. See, Grace et al., J Biol Chem.279:8102-8110 (2004); and Shirsat et al., supra. A regulatory region cancontain a TATABOXOSPAL motif having the consensus sequence TATTTAA. See,Zhu et al., Plant Cell 14: 795-803 (2002). A regulatory region cancontain a TATCCAYMOTIFOSRAMY3D motif having the consensus sequenceTATCCAY. See, Toyofuku et al., FEBS Lett 428:275-280 (1998); andRubio-Somoza et al., Plant J. 47: 269-281 (2006). A regulatory regioncan contain a TE2F2NTPCNA motif having the consensus sequence ATTCCCGC.See, Kosugi and Ohashi, Plant J 29: 45-59 (2002). A regulatory regioncan contain a TRANSINITMONOCOTS motif having the consensus sequenceRMNAUGGC. See, Joshi et al., Plant Mol Biol 35:993-1001 (1997). Aregulatory region can contain a UP2ATMSD motif having the consensussequence AAACCCTA. See, Tatematsu et al., Plant Physiol. 138: 757-766(2005). A regulatory region can contain an UPRMOTIFIIAT motif having theconsensus sequence CCNNNNNNNNNNNNCCACG (SEQ ID NO:26). See, Martinez andChrispeels, Plant Cell. 15:561-576 (2003); and Oh et al., BiochemBiophys Res Commun. 301:225-230 (2003).

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:1 or a fragment thereof, wherein the nucleic acidcontains a GARE1OSREP1, ACIIPVPAL2, ABRERATCAL, and TATABOX motif. TheGARE1OSREP1 motif can be the motif at nucleotides 80 to 86 ornucleotides 122 to 128 of SEQ ID NO:1 or a GARE1OSREP1 motifheterologous to those in SEQ ID NO:1. The ACIIPVPAL2 motif can be themotif at nucleotides 201 to 212 of SEQ ID NO:1 or an ACIIPVPAL2 motifheterologous to that in SEQ ID NO:1. The ABRERATCAL motif can be themotif at nucleotides 327 to 333 of SEQ ID NO:1 or an ABRERATCAL motifheterologous to that in SEQ ID NO:1. The TATABOX can be the motif atnucleotides 461 to 467 of SEQ ID NO:1 or a TATABOX heterologous to thatin SEQ ID NO:1. In some cases, such a regulatory region can also includea 5′ UTR. The 5′ UTR can be the 5′ UTR at nucleotides 501 to 561 or 1069to 1100 of SEQ ID NO:1 or can be a heterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:2 or a fragment thereof, wherein the nucleic acidcontains an AUXRETGA2GMGH3, ACGTABREMOTIFA2OSEM, TATCCAYMOTIFOSRAMY3D,AMYBOX1, and GAREAT motif The AUXRETGA2GMGH3 motif can be the motif atnucleotides 24 to 32 of SEQ ID NO:2 or an AUXRETGA2GMGH3 motifheterologous to that in SEQ ID NO:2. The ACGTABREMOTIFA2OSEM motif canbe the motif at nucleotides 24 to 30 of SEQ ID NO:2 or anACGTABREMOTIFA2OSEM motif heterologous to that in SEQ ID NO:2. TheTATCCAYMOTIFOSRAMY3D motif can be the motif at nucleotides 52 to 58 ofSEQ ID NO:2 or a TATCCAYMOTIFOSRAMY3D motif heterologous to that in SEQID NO:2. The AMYBOX1 motif can be the motif at nucleotides 135 to 141 ofSEQ ID NO:2 or an AMYBOX1 motif heterologous to that in SEQ ID NO:2. TheGAREAT motif can be the motif at nucleotides 135 to 141 of SEQ ID NO:2or a GAREAT motif heterologous to that in SEQ ID NO:1. In some cases,such a regulatory region can also include a 5′ UTR. The 5′ UTR can bethe 5′ UTR at nucleotides 140 to 219 of SEQ ID NO:2 or can be aheterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:3 or a fragment thereof, wherein the nucleic acidcontains an ARE1, SBOXATRBCS, TE2F2NTPCNA, GADOWNAT,ACGTABREMOTIFA2OSEM, and TATABOX motif. The ARE1 motif can be the motifat nucleotides 124 to 134 of SEQ ID NO:3 or an ARE1 motif heterologousto that in SEQ ID NO:3. The SBOXATRBCS motif can be the motif atnucleotides 202 to 209 of SEQ ID NO:3 or a SBOXATRBCS motif heterologousto that in SEQ ID NO:3. The TE2F2NTPCNA motif can be the motif atnucleotides 240 to 247 of SEQ ID NO:3 or a TE2F2NTPCNA motifheterologous to that in SEQ ID NO:3. The GADOWNAT motif can be the motifat nucleotides 308 to 314 of SEQ ID NO:3 or a GADOWNAT motifheterologous to that in SEQ ID NO:3. The ACGTABREMOTIFA2OSEM motif canbe the motif at nucleotides 308 to 314 of SEQ ID NO:3 or anACGTABREMOTIFA2OSEM motif heterologous to that in SEQ ID NO:3. TheTATABOX can be the motif at nucleotides 280 to 286 of SEQ ID NO:3 or aTATABOX heterologous to that in SEQ ID NO:3. In some cases, such aregulatory region can also include a 5′ UTR. The 5′ UTR can be the 5′UTR at nucleotides 311 to 400 of SEQ ID NO:3 or can be a heterologousUTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:4 or a fragment thereof, wherein the nucleic acidcontains a ROOTMOTIFTAPOX1, RYREPEATVFLEB4, and TATABOX motif. TheROOTMOTIFTAPOX1 motif can be the motif at nucleotides 179 to 183 or 182to 186 of SEQ ID NO:4 or a ROOTMOTIFTAPDX1 motif heterologous to thosein SEQ ID NO:4. The RYREPEATVFLEB4 motif can be the motif at nucleotides225 to 232 or 229 to 236 of SEQ ID NO:4 or a RYREPEATVFLEB4 motifheterologous to those in SEQ ID NO:4. The TATABOX can be the motif atnucleotides 292 to 299 of SEQ ID NO:4 or a TATABOX motif heterologous tothat in SEQ ID NO:4. In some cases, such a regulatory region can alsoinclude a 5′ UTR. The 5′ UTR can be the 5′ UTR at nucleotides 324 to 420of SEQ ID NO:4 or can be a heterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:5 or a fragment thereof, wherein the nucleic acidcontains a CARGCW8GAT, INRNTPSADB, and TATABOX2motif. The CARGCW8GATmotif can be the motif at nucleotides 415 to 424 of SEQ ID NO:5 or aCARGCW8GAT motif heterologous to that in SEQ ID NO:5. The INRNTPSADBmotif can be the motif at nucleotides 580 to 587 of SEQ ID NO:5 or anINRNTPSADB motif heterologous to that in SEQ ID NO:5. The TATABOX2 motifcan be the motif at nucleotides 417 to 423 of SEQ ID NO:5 or a TATABOX2motif heterologous to that in SEQ ID NO:5. In some cases, such aregulatory region can also include a 5′ UTR. The 5′ UTR can be the 5′UTR at nucleotides 630 to 759 of SEQ ID NO:5 or can be a heterologousUTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:6 or a fragment thereof, wherein the nucleic acidcontains a MARTBOX, RYREPEATVFLEB4, NRRBNEXTA, TRANSINITMONOCOTS,TATABOXOSPAL, and P1BS motif. The MARTBOX motif can be the motif atnucleotides 453 to 462 of SEQ ID NO:6 or a MARTBOX motif heterologous tothat in SEQ ID NO:6. The RYREPEATVFLEB4 motif can be the motif atnucleotides 480 to 487 or 832 to 839 of SEQ ID NO:6 or a RYREPEATVFLEB4motif heterologous to those in SEQ ID NO:6. The NRRBNEXTA motif can bethe motif at nucleotides 693 to 700 of SEQ ID NO:6 or a NRRBNEXTA motifheterologous to that in SEQ ID NO:6. The TRANSINITMONOCOTS motif can bethe motif at nucleotides 853 to 860 of SEQ ID NO:6 or aTRANSINITMONOCOTS motif heterologous to that in SEQ ID NO:6. TheTATABOXOSPAL motif can be the motif at nucleotides 884 to 890 of SEQ IDNO:6 or a TATABOXOSPAL motif heterologous to that in SEQ ID NO:6. TheP1BS motif can be the motif at nucleotides 907 to 914 of SEQ ID NO:6 ora P1BS motif heterologous to that in SEQ ID NO:6. In some cases, such aregulatory region can also include a 5′ UTR. The 5′ UTR can be the 5′UTR at nucleotides 914 to 1020 of SEQ ID NO:6 or can be a heterologousUTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:7 or a fragment thereof, wherein the nucleic acidcontains a CARGCW8GAT, P1BS, and TATABOX motif. The CARGCW8GAT motif canbe the motif at nucleotides 294 to 303 or 394 to 403 of SEQ ID NO:7 or aCARGCW8GAT motif heterologous to those in SEQ ID NO:7. The P1BS motifcan be the motif at nucleotides 666 to 673 of SEQ ID NO:7 or a P1BSmotif heterologous to that in SEQ ID NO:7. The TATABOX can be the motifat nucleotides 883 to 891 of SEQ ID NO:7 or a TATABOX motif heterologousto that in SEQ ID NO:7. In some cases, such a regulatory region can alsoinclude a 5′ UTR. The 5′ UTR can be the 5′ UTR at nucleotides 917 to1075 of SEQ ID NO:7 or can be a heterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:8 or a fragment thereof, wherein the nucleic acidcontains a ROOTMOTIFTAPOX1, ATHB6COREAT, TATABOX2, and UP2ATMSD motif.The ROOTMOTIFTAPOX1 motif can be the motif at nucleotides 120 to 124,392 to 396, or 522 to 526 of SEQ ID NO:8 or a ROOTMOTIFTAPOX1 motifheterologous to those in SEQ ID NO:8. The ATHB6COREAT motif can be themotif at nucleotides 779 to 787 of SEQ ID NO:8 or an ATHB6COREAT motifheterologous to that in SEQ ID NO:8. The TATABOX2 motif can be the motifat nucleotides 813 to 819 of SEQ ID NO:8 or a TATABOX2 motifheterologous to that in SEQ ID NO:8. The UP2ATMSD motif can be the motifat nucleotides 951 to 958 of SEQ ID NO:8 or an UP2ATMSD motifheterologous to that in SEQ ID NO:8. In some cases, such a regulatoryregion can also include a 5′ UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:9 or a fragment thereof, wherein the nucleic acidcontains a PRECONSCRHSP70A, IBOXCORENT, AGCBOXNPGLB, UP2ATMSD, andTATABOX4 motif. The PRECONSCRHSP70A motif can be the motif atnucleotides 22 to 45, 38 to 61, or 60 to 83 of SEQ ID NO:9 or aPRECONSCRHSP70A motif heterologous to those in SEQ ID NO:9. TheIBOXCORENT motif can be the motif at nucleotides 153 to 159 of SEQ IDNO:9 or an IBOXCORENT motif heterologous to that in SEQ ID NO:9. TheAGCBOXNPGLB motif can be the motif at nucleotides 216 to 222 or 306 to312 of SEQ ID NO:9 or an AGCBOXNPGLB motif heterologous to those in SEQID NO:9. The UP2ATMSD motif can be the motif at nucleotides 306 to 312of SEQ ID NO:9 or an UP2ATMSD motif heterologous to that in SEQ ID NO:9.The TATABOX4 motif can be the motif at nucleotides 359 to 365 of SEQ IDNO:9 or a TATABOX4 motif heterologous to that in SEQ ID NO:9. In somecases, such a regulatory region can also include a 5′ UTR. The 5′ UTRcan be the 5′ UTR at nucleotides 390 to 1428 of SEQ ID NO:9 or can be aheterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:10 or a fragment thereof, wherein the nucleic acidcontains an IBOXCORENT, ERELEE4, ABREATRD22, P1BS, and TATABOX motif.The IBOXCORENT motif can be the motif at nucleotides 1472 to 1478 of SEQID NO:10 or an IBOXCORENT motif heterologous to that in SEQ ID NO:10.The ERELEE4 motif can be the motif at nucleotides 1565 to 1572 or 2270to 2277 of SEQ ID NO:10 or an ERELEE4 motif heterologous to those in SEQID NO:10. The ABREATRD22 motif can be the motif at nucleotides 2193 to2202 of SEQ ID NO:10 or an ABREATRD22 motif heterologous to that in SEQID NO:10. The P1BS motif can be the motif at nucleotides 2353 to 2360 ofSEQ ID NO:10 or a P1BS motif heterologous to that in SEQ ID NO:10. TheTATABOX motif can be the motif at nucleotides 2391 to 2396 of SEQ IDNO:10 or a TATABOX motif heterologous to that in SEQ ID NO:10. In somecases, such a regulatory region can also include a 5′ UTR. The 5′ UTRcan be the 5′ UTR at nucleotides 2426 to 2485 of SEQ ID NO:10 or can bea heterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:11 or a fragment thereof (e.g., a fragment containingnucleotides 1 to 896 of SEQ ID NO:11), wherein the nucleic acid containsa CACGCAATGMGH3 and UPRMOTIFIIAT motif. The CACGCAATGMGH3 motif can bethe motif at nucleotides 716 to 723 of SEQ ID NO:11 or a CACGCAATGMGH3motif heterologous to that in SEQ ID NO:11. The UPRMOTIFIIAT motif canbe the motif at nucleotides 770 to 788 of SEQ ID NO:11 or anUPRMOTIFIIAT motif heterologous to that in SEQ ID NO:11. In some cases,such a regulatory region can also include a 5′ UTR. The 5′ UTR can bethe 5′ UTR at nucleotides 837 to 896 of SEQ ID NO:11 or can be aheterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:12 or a fragment thereof (e.g., a fragment containingnucleotides 501 to 2000 of SEQ ID NO:12, referred to as PD3777 herein),wherein the nucleic acid contains a PRECONSCRHSP70A, ACIIPVPAL2,TATABOX4, and CAAT-box motif. The PRECONSCRHSP70A motif can be the motifat nucleotides 535 to 558 of SEQ ID NO:12 or a PRECONSCRHSP70A motifheterologous to that in SEQ ID NO:12. The ACIIPVPAL2 motif can be themotif at nucleotides 765 to 776 of SEQ ID NO:12 or an ACIIPVPAL2 motifheterologous to that in SEQ ID NO:12. The TATABOX4 motif can be themotif at nucleotides 954 to 960 of SEQ ID NO:12 or a TATABOX4 motifheterologous to that in SEQ ID NO:12. The CAAT-box motif can be themotif at nucleotides 986 to 990 of SEQ ID NO:12 or a CAAT-box motifheterologous to that in SEQ ID NO:12. In some embodiments, suchregulatory region also contains a CAATBOX1 and UPRMOTIFIIAT motif. TheCAATBOX1 motif can be the motif at nucleotides 11 to 15 of SEQ ID NO:12or a CAATBOX1 motif heterologous to that in SEQ ID NO:12. TheUPRMOTIFIIAT motif can be the motif at nucleotides 326 to 344 of SEQ IDNO:12 or an UPRMOTIFIIAT motif heterologous to that in SEQ ID NO:12. Insome cases, such regulatory regions can also include a 5′ UTR. The 5′UTR can be the 5′ UTR at nucleotides 984 to 1043 of SEQ ID NO:12 or canbe a heterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:13 or a fragment thereof, wherein the nucleic acidcontains a RYREPEATVFLEB4, SPHCOREZMC1, and AGCBOXNPGLB motif. TheRYREPEATVFLEB4 motif can be the motif at nucleotides 786 to 793 of SEQID NO:13 or a RYREPEATVFLEB4 motif heterologous to that in SEQ ID NO:13.The SPHCOREZMC1 motif can be the motif at nucleotides 787 to 795 of SEQID NO:13 or a SPHCOREZMC1 motif heterologous to that in SEQ ID NO:13.The AGCBOXNPGLB motif can be the motif at nucleotides 1082 to 1088 ofSEQ ID NO:13 or an AGCBOXNPGLB motif heterologous to that in SEQ IDNO:13. In some cases, such a regulatory region can also include a 5′UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:14 or a fragment thereof (e.g., nucleotides 501 to990 of SEQ ID NO:14), wherein the nucleic acid contains aRYREPEATVFLEB4, P1BS, and TATABOX1 motif. The RYREPEATVFLEB4 motif canbe the motif at nucleotides 707 to 713 or 795 to 801 of SEQ ID NO:14 ora RYREPEATVFLEB4 motif heterologous to those in SEQ ID NO:14. The P1BSmotif can be the motif at nucleotides 798 to 805 of SEQ ID NO:14 or aP1BS motif heterologous to that in SEQ ID NO:14. The TATABOX1 motif canbe the motif at nucleotides 855 to 864 of SEQ ID NO:14 or a TATABOX1motif heterologous to that in SEQ ID NO:14. In some embodiments, such aregulatory region also includes a ROOTMOTIFTAPOX1 motif. For example,the ROOTMOTIFTAPOX1 motif can be the motif at nucleotides 27 to 31, 77to 81, or 145 to 149 of SEQ ID NO:14 or a ROOTMOTIFTAPOX1 motifheterologous to those in SEQ ID NO:14. In some cases, such regulatoryregions can also include a 5′ UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:15 or a fragment thereof (e.g., nucleotides 702 to1500 of SEQ ID NO:15), wherein the nucleic acid contains a SBOXATRBCS,MYBbindingsite, and ATHB1ATCONSENSUS motif. The SBOXATRBCS motif can bethe motif at nucleotides 846 to 853 of SEQ ID NO:15 or a SBOXATRBCSmotif heterologous to those in SEQ ID NO:15. The MYBbindingsite motifcan be the motif at nucleotides 994 to 999 of SEQ ID NO:15 or aMYBbindingsite motif heterologous to that in SEQ ID NO:15. TheATHB1ATCONSENSUS motif can be the motif at nucleotides 1315 to 1323 ofSEQ ID NO:15 or a ATHB1ATCONSENSUS motif heterologous to that in SEQ IDNO:15. In some embodiments, such a regulatory region also includes anABRE, E2FAT, PRECONSCRHSP70A, MYBPLANT, and E2FCONSENSUS motif. Forexample, the ABRE motif can be the motif at nucleotides 80 to 85 of SEQID NO:15 or an ABRE motif heterologous to those in SEQ ID NO:15. TheE2FAT motif can be the motif at nucleotides 139 to 147 of SEQ ID NO:15or an E2FAT motif heterologous to that in SEQ ID NO:15. ThePRECONSCRHSP70A motif can be the motif at nucleotides 167 to 190 or 297to 320 of SEQ ID NO:15 or a PRECONSCRHSP70A motif heterologous to thosein SEQ ID NO:15. The MYBPLANT motif can be the motif at nucleotides 269to 276 of SEQ ID NO:15 or a MYBPLANT motif heterologous to that in SEQID NO:15. The E2FCONSENSUS motif can be the motif at nucleotides 526 to533 of SEQ ID NO:15 or a E2FCONSENSUS motif heterologous to that in SEQID NO:15. In some cases, such regulatory regions can also include a 5′UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:16 or a fragment thereof, wherein the nucleic acidcontains a P1BS, ABREZMRAB28, SP8BFIBSP8BIB, CCA1ATLHCB1, BOXIIPCCHS,and LRENPCABE motif. The P1BS motif can be the motif at nucleotides 739to 746 of SEQ ID NO:16 or a P1BS motif heterologous to that in SEQ IDNO:16. The ABREZMRAB2 motif can be the motif at nucleotides 349 to 356of SEQ ID NO:16 or an ABREZMRAB2 motif heterologous to that in SEQ IDNO:16. The SP8BFIBSP8BIB motif can be the motif at nucleotides 1168 to1174, 1347 to 1353, or 1377 to 1383 of SEQ ID NO:16 or an SP8BFIBSP8BIBmotif heterologous to those in SEQ ID NO:16. The CCA1ATLHCB1 motif canbe the motif at nucleotides 1509 to 1516 of SEQ ID NO:16 or aCCA1ATLHCB1 motif heterologous to that in SEQ ID NO:16. The BOXIIPCCHSmotif can be the motif at nucleotides 1624 to 1630 of SEQ ID NO:16 or aBOXIIPCCHS motif heterologous to that in SEQ ID NO:16. The LRENPCABEmotif can be the motif at nucleotides 1624 to 1631 of SEQ ID NO:16 or aLRENPCABE motif heterologous to that in SEQ ID NO:16. In some cases,such a regulatory region can also include a 5′ UTR. The 5′ UTR can bethe 5′ UTR at nucleotides 1857 to 1989 of SEQ ID NO:16 or can be aheterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:17 or a fragment thereof, wherein the nucleic acidcontains a P1BS, MYBGAHV, and CEREGLUBOX2PSLEGA motif. The P1BS motifcan be the motif at nucleotides 261 to 268 or 716 to 723 of SEQ ID NO:17or a P1BS motif heterologous to that in SEQ ID NO:17. The MYBGAHV motifcan be the motif at nucleotides 430 to 436 of SEQ ID NO:17 or a MYBGAHVmotif heterologous to that in SEQ ID NO:17. The CEREGLUBOX2PSLEGA motifcan be the motif at nucleotides 484 to 491 of SEQ ID NO:17 or aCEREGLUBOX2PSLEGA motif heterologous to that in SEQ ID NO:17. In somecases, such a regulatory region can also include a 5′ UTR. The 5′ UTRcan be the 5′ UTR at nucleotides 930 to 1000 of SEQ ID NO:17 or can be aheterologous UTR.

In some embodiments, a regulatory region has a nucleotide sequence with90% or greater sequence identity to the polynucleotide sequence setforth in SEQ ID NO:18 or a fragment thereof (nucleotides 353 to 1248 ofSEQ ID NO:18), wherein the nucleic acid contains a CACGCAATGMGH3 orUPRMOTIFIIAT motif. It is noted that nucleotides 353 to 1500 of SEQ IDNO:18 are identical to nucleotides 1 to 1148 of SEQ ID NO:11. TheCACGCAATGMGH3 motif can be the motif at nucleotides 1068 to 1075 of SEQID NO:18 or a CACGCAATGMGH3 motif heterologous to that in SEQ ID NO:18.The UPRMOTIFIIA motif can be the motif at nucleotides 1122 to 1140 ofSEQ ID NO:18 or an UPRMOTIFIIA motif heterologous to that in SEQ IDNO:18. In some cases, such a regulatory region can also include a 5′UTR. The 5′ UTR can be the 5′ UTR at nucleotides 1189 to 1248 of SEQ IDNO:18 or can be a heterologous UTR.

4. TESTING OF PROMOTERS

Promoters of the document were tested for activity by cloning thesequence into an appropriate vector, transforming plants with theconstruct and assaying for marker gene expression. Recombinant DNAconstructs were prepared which comprise the promoter sequences of thedocument inserted into a vector suitable for transformation of plantcells. The construct can be made using standard recombinant DNAtechniques (Sambrook et al. 1989) and can be introduced to the speciesof interest by Agrobacterium-mediated transformation or by other meansof transformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

-   (a) BAC: Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89:    8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA 93:    9975-9979;-   (b) YAC: Burke et al. (1987) Science 236:806-812;-   (c) PAC: Sternberg N. et al. (1990) Proc Natl Acad Sci USA.    87(1):103-7;-   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al. (1995) Nucl    Acids Res 23: 4850-4856;-   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et    al. (1983) J. Mol Biol 170: 827-842; or Insertion vector, e.g.,    Huynh et al. (1985) In: Glover N. Mex. (ed) DNA Cloning: A practical    Approach, Vol. 1 Oxford: IRL Press; T-DNA gene fusion vectors:    Walden et al. (1990) Mol Cell Biol 1: 175-194; and-   (g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a promotersequence of the present document operationally linked to any markergene. The promoter was identified as a promoter by the expression of themarker gene. Although many marker genes can be used, Green FluorescentProtein (GFP) is preferred. The vector may also comprise a marker genethat confers a selectable phenotype on plant cells. The marker mayencode biocide resistance, particularly antibiotic resistance, such asresistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or phosphinothricin.Vectors can also include origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, introns, etc.

5. CONSTRUCTING PROMOTERS WITH CONTROL ELEMENTS

5.1 Combining Promoters and Promoter Control Elements

The promoter and promoter control elements of the present document, bothnaturally occurring and synthetic, can be used alone or combined witheach other to produce the desired preferential transcription. Also, thepromoters of the document can be combined with other known sequences toobtain other useful promoters to modulate, for example, tissuetranscription specific or transcription specific to certain conditions.Such preferential transcription can be determined using the techniquesor assays described above.

Promoters can contain any number of control elements. For example, apromoter can contain multiple transcription binding sites or othercontrol elements. One element may confer tissue or organ specificity;another element may limit transcription to specific time periods, etc.Typically, promoters will contain at least a basal or core promoter asdescribed above. Any additional element can be included as desired. Forexample, a fragment comprising a basal or “core” promoter can be fusedwith another fragment with any number of additional control elements.

The following are promoters that are induced under stress conditions andcan be combined with those of the present document: 1dh1 (oxygen stress;tomato; see Germain and Ricard (1997) Plant Mol Biol 35:949-54), GPx andCAT (oxygen stress; mouse; see Franco et al. (1999) Free Radic Biol Med27:1122-32), ci7 (cold stress; potato; see Kirch et al. (1997) Plant MolBiol. 33:897-909), Bz2 (heavy metals; maize; see Marrs and Walbot (1997)Plant Physiol 113:93-102), HSP32 (hyperthermia; rat; see Raju and Maines(1994) Biochim Biophys Acta 1217:273-80), and MAPKAPK-2 (heat shock;Drosophila; see Larochelle and Suter (1995) Gene 163:209-14).

In addition, the following examples of promoters are induced by thepresence or absence of light can be used in combination with those ofthe present document: Topoisomerase II (pea; see Reddy et al. (1999)Plant Mol Biol 41:125-37), chalcone synthase (soybean; see Wingender etal. (1989) Mol Gen Genet 218:315-22) mdm2 gene (human tumor; see Saucedoet al. (1998) Cell Growth Differ 9:119-30), Clock and BMAL1 (rat; seeNamihira et al. (1999) Neurosci Lett 271:1-4, PHYA (Arabidopsis; seeCanton and Quail (1999) Plant Physiol 121:1207-16), PRB-1b (tobacco; seeSessa et al. (1995) Plant Mol Biol 28:537-47) and Ypr10 (common bean;see Walter et al. (1996) Eur J Biochem 239:281-93).

The promoters and control elements of the following genes can be used incombination with the present document to confer tissue specificity: MipB(iceplant; Yamada et al. (1995) Plant Cell 7:1129-42) and SUCS (rootnodules; broadbean; Kuster et al. (1993) Mol Plant Microbe Interact6:507-14) for roots, OsSUT1 (rice; Hirose et al. (1997) Plant CellPhysiol 38:1389-96) for leaves, Msg (soybean; Stomvik et al. (1999)Plant Mol Biol 41:217-31) for siliques, cell (Arabidopsis; Shani et al.(1997) Plant Mol Biol 34(6):837-42) and ACT11 (Arabidopsis; Huang et al.(1997) Plant Mol Biol 33:125-39) for inflorescence.

Still other promoters are affected by hormones or participate inspecific physiological processes, which can be used in combination withthose of present document. Some examples are the ACC synthase gene thatis induced differently by ethylene and brassinosteroids (mung bean; Yiet al. (1999) Plant Mol Biol 41:443-54), the TAPG1 gene that is activeduring abscission (tomato; Kalaitzis et al. (1995) Plant Mol Biol28:647-56), and the 1-aminocyclopropane-1-carboxylate synthase gene(carnation; Jones et al. (1995) Plant Mol Biol 28:505-12) and theCP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol Reprod57:1467-77), both active during senescence.

Spacing between control elements or the configuration or controlelements can be determined or optimized to permit the desiredprotein-polynucleotide or polynucleotide interactions to occur.

For example, if two transcription factors bind to a promotersimultaneously or relatively close in time, the binding sites are spacedto allow each factor to bind without steric hindrance. The spacingbetween two such hybridizing control elements can be as small as aprofile of a protein bound to a control element. In some cases, twoprotein binding sites can be adjacent to each other when the proteinsbind at different times during the transcription process.

Further, when two control elements hybridize the spacing between suchelements will be sufficient to allow the promoter polynucleotide tohairpin or loop to permit the two elements to bind. The spacing betweentwo such hybridizing control elements can be as small as a t-RNA loop,to as large as 10 kb.

Typically, the spacing is no smaller than 5 bases; more typically, nosmaller than 8; more typically, no smaller than 15 bases; moretypically, no smaller than 20 bases; more typically, no smaller than 25bases; even more typically, no smaller than 30, 35, 40 or 50 bases.

Usually, the fragment size in no larger than 5 kb bases; more usually,no larger than 2 kb; more usually, no larger than 1 kb; more usually, nolarger than 800 bases; more usually, no larger than 500 bases; even moreusually, no more than 250, 200, 150 or 100 bases.

Such spacing between promoter control elements can be determined usingthe techniques and assays described above.

5.2 Vectors Used to Transform Cells/Hosts

A plant transformation construct containing a promoter of the presentdocument may be introduced into plants by any plant transformationmethod. Methods and materials for transforming plants by introducing aplant expression construct into a plant genome in the practice of thisdocument can include any of the well-known and demonstrated methodsincluding electroporation (U.S. Pat. No. 5,384,253); microprojectilebombardment (U.S. Pat. No. 5,015,580; U.S. Pat. No. 5,550,318; U.S. Pat.No. 5,538,880; U.S. Pat. No. 6,160,208; U.S. Pat. No. 6,399,861; andU.S. Pat. No. 6,403,865); Agrobacterium-mediated transformation (U.S.Pat. No. 5,824,877; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,981,840;and U.S. Pat. No. 6,384,301); and protoplast transformation (U.S. Pat.No. 5,508,184).

The present promoters and/or promoter control elements may be deliveredto a system such as a cell by way of a vector. For the purposes of thisdocument, such delivery may range from simply introducing the promoteror promoter control element by itself randomly into a cell tointegration of a cloning vector containing the present promoter orpromoter control element. Thus, a vector need not be limited to a DNAmolecule such as a plasmid, cosmid or bacterial phage that has thecapability of replicating autonomously in a host cell. All other mannerof delivery of the promoters and promoter control elements of thedocument are envisioned. The various T-DNA vector types are a preferredvector for use with the present document. Many useful vectors arecommercially available.

It may also be useful to attach a marker sequence to the presentpromoter and promoter control element in order to determine activity ofsuch sequences. Marker sequences typically include genes that provideantibiotic resistance, such as tetracycline resistance, hygromycinresistance or ampicillin resistance, or provide herbicide resistance.Specific selectable marker genes may be used to confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai et al.(1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; and Stalker et al. (1988) Science 242: 419-423). Other markergenes exist which provide hormone responsiveness.

The promoter or promoter control element of the present document may beoperably linked to a polynucleotide to be transcribed. In this manner,the promoter or promoter control element may modify transcription bymodulating transcript levels of that polynucleotide when inserted into agenome.

However, prior to insertion into a genome, the promoter or promotercontrol element need not be linked, operably or otherwise, to apolynucleotide to be transcribed. For example, the promoter or promotercontrol element may be inserted alone into the genome in front of apolynucleotide already present in the genome. In this manner, thepromoter or promoter control element may modulate the transcription of apolynucleotide that was already present in the genome. Thispolynucleotide may be native to the genome or inserted at an earliertime.

Alternatively, the promoter or promoter control element may be insertedinto a genome alone to modulate transcription. See, for example,Vaucheret, H et al. (1998) Plant J 16: 651-659. Rather, the promoter orpromoter control element may be simply inserted into a genome ormaintained extrachromosomally as a way to divert transcription resourcesof the system to itself. This approach may be used to downregulate thetranscript levels of a group of polynucleotide(s).

The nature of the polynucleotide to be transcribed is not limited.Specifically, the polynucleotide may include sequences that will haveactivity as RNA as well as sequences that result in a polypeptideproduct. These sequences may include, but are not limited to antisensesequences, RNAi sequences, ribozyme sequences, spliceosomes, amino acidcoding sequences, and fragments thereof. Specific coding sequences mayinclude, but are not limited to endogenous proteins or fragmentsthereof, or heterologous proteins including marker genes or fragmentsthereof.

Constructs of the present document would typically contain a promoteroperably linked to a transcribable nucleic acid molecule operably linkedto a 3′ transcription termination nucleic acid molecule. In addition,constructs may include but are not limited to additional regulatorynucleic acid molecules from the 3′-untranslated region (3′ UTR) of plantgenes (e.g., a 3′ UTR to increase mRNA stability of the mRNA, such asthe PI-II termination region of potato or the octopine or nopalinesynthase 3′ termination regions). Constructs may include but are notlimited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acidmolecule which can play an important role in translation initiation andcan also be a genetic component in a plant expression construct. Forexample, non-translated 5′ leader nucleic acid molecules derived fromheat shock protein genes have been demonstrated to enhance geneexpression in plants (see for example, U.S. Pat. No. 5,659,122 and U.S.Pat. No. 5,362,865, all of which are hereby incorporated by reference).These additional upstream and downstream regulatory nucleic acidmolecules may be derived from a source that is native or heterologouswith respect to the other elements present on the promoter construct.

Thus, one embodiment of the document is a promoter such as provided inSEQ ID NOs: 1-18 or a fragment thereof, operably linked to atranscribable nucleic acid molecule so as to direct transcription ofsaid transcribable nucleic acid molecule at a desired level or in adesired tissue or developmental pattern upon introduction of saidconstruct into a plant cell. In some cases, the transcribable nucleicacid molecule comprises a protein-coding region of a gene, and thepromoter provides for transcription of a functional mRNA molecule thatis translated and expressed as a protein product. Constructs may also beconstructed for transcription of antisense RNA molecules or othersimilar inhibitory RNA in order to inhibit expression of a specific RNAmolecule of interest in a target host cell.

Exemplary transcribable nucleic acid molecules for incorporation intoconstructs of the present document include, for example, nucleic acidmolecules or genes from a species other than the target gene species, oreven genes that originate with or are present in the same species, butare incorporated into recipient cells by genetic engineering methodsrather than classical reproduction or breeding techniques. Exogenousgene or genetic element is intended to refer to any gene or nucleic acidmolecule that is introduced into a recipient cell. The type of nucleicacid molecule included in the exogenous nucleic acid molecule caninclude a nucleic acid molecule that is already present in the plantcell, a nucleic acid molecule from another plant, a nucleic acidmolecule from a different organism, or a nucleic acid molecule generatedexternally, such as a nucleic acid molecule containing an antisensemessage of a gene, or a nucleic acid molecule encoding an artificial ormodified version of a gene.

The promoters of the present document can be incorporated into aconstruct using marker genes as described, and tested in transientanalyses that provide an indication of gene expression in stable plantsystems. As used herein the term “marker gene” refers to anytranscribable nucleic acid molecule whose expression can be screened foror scored in some way. Methods of testing for marker gene expression intransient assays are known to those of skill in the art. Transientexpression of marker genes has been reported using a variety of plants,tissues, plant cell(s), and DNA delivery systems. For example, types oftransient analyses can include but are not limited to direct genedelivery via electroporation or particle bombardment of tissues in anytransient plant assay using any plant species of interest. Suchtransient systems would include, but are not limited to, electroporationof protoplasts from a variety of tissue sources or particle bombardmentof specific tissues of interest. The present document encompasses theuse of any transient expression system to evaluate promoters or promoterfragments operably linked to any transcribable nucleic acid molecules,including but not limited to selected reporter genes, marker genes, orgenes of agronomic interest. Examples of plant tissues envisioned totest in transients via an appropriate delivery system would include, butare not limited to, leaf base tissues, callus, cotyledons, roots,endosperm, embryos, floral tissue, pollen, and epidermal tissue.

Promoters and control elements of the present document are useful formodulating metabolic or catabolic processes. Such processes include, butare not limited to, secondary product metabolism, amino acid synthesis,seed protein storage, increased biomass, oil development, pest defenseand nitrogen usage. Some examples of genes, transcripts and peptides orpolypeptides participating in these processes, which can be modulated bythe present document: are tryptophan decarboxylase (tdc) andstrictosidine synthase (str1), dihydrodipicolinate synthase (DHDPS) andaspartate kinase (AK), 2S albumin and alpha-, beta-, and gamma-zeins,ricinoleate and 3-ketoacyl-ACP synthase (KAS), Bacillus thuringiensis(Bt) insecticidal protein, cowpea trypsin inhibitor (CpTI), asparaginesynthetase and nitrite reductase. Alternatively, expression constructscan be used to inhibit expression of these peptides and polypeptides byincorporating the promoters in constructs for antisense use,co-suppression use or for the production of dominant negative mutations.

As explained above, several types of regulatory elements existconcerning transcription regulation. Each of these regulatory elementsmay be combined with the present vector if desired. Translation ofeukaryotic mRNA is often initiated at the codon that encodes the firstmethionine. Thus, when constructing a recombinant polynucleotideaccording to the present document for expressing a protein product, itis preferable to ensure that the linkage between the 3′ portion,preferably including the TATA box, of the promoter and thepolynucleotide to be transcribed, or a functional derivative thereof,does not contain any intervening codons which are capable of encoding amethionine.

The vector of the present document may contain additional components.For example, an origin of replication allows for replication of thevector in a host cell. Additionally, homologous sequences flanking aspecific sequence allow for specific recombination of the specificsequence at a desired location in the target genome. T-DNA sequencesalso allow for insertion of a specific sequence randomly into a targetgenome.

The vector may also be provided with a plurality of restriction sitesfor insertion of a polynucleotide to be transcribed as well as thepromoter and/or promoter control elements of the present document. Thevector may additionally contain selectable marker genes. The vector mayalso contain a transcriptional and translational initiation region, anda transcriptional and translational termination region functional in thehost cell. The termination region may be native with the transcriptionalinitiation region, may be native with the polynucleotide to betranscribed, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al.(1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the polynucleotide to be transcribed may be optimizedfor increased expression in a certain host cell. For example, thepolynucleotide can be synthesized using preferred codons for improvedtranscription and translation. See U.S. Pat. Nos. 5,380,831, 5,436,391;see also and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

Additional sequence modifications include elimination of sequencesencoding spurious polyadenylation signals, exon intron splice sitesignals, transposon-like repeats, and other such sequences wellcharacterized as deleterious to expression. The G-C content of thepolynucleotide may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. The polynucleotide sequence may be modified to avoid hairpinsecondary mRNA structures.

A general description of expression vectors and reporter genes can befound in Gruber, et al. (1993) “Vectors for Plant Transformation” InMethods in Plant Molecular Biology & Biotechnology, Glich et al. Eds.pp. 89-119, CRC Press. Moreover GUS expression vectors and GUS genecassettes are available from Clonetech Laboratories, Inc., Palo Alto,Calif. while luciferase expression vectors and luciferase gene cassettesare available from Promega Corp. (Madison, Wis.). GFP vectors areavailable from Aurora Biosciences.

5.3 Polynucleotide Insertion into a Host Cell

The promoters according to the present document can be inserted into ahost cell. A host cell includes but is not limited to a plant,mammalian, insect, yeast, and prokaryotic cell, preferably a plant cell.

The method of insertion into the host cell genome is chosen based onconvenience. For example, the insertion into the host cell genome mayeither be accomplished by vectors that integrate into the host cellgenome or by vectors which exist independent of the host cell genome.

The promoters of the present document can exist autonomously orindependent of the host cell genome. Vectors of these types are known inthe art and include, for example, certain type of non-integrating viralvectors, autonomously replicating plasmids, artificial chromosomes, andthe like.

Additionally, in some cases transient expression of a promoter may bedesired.

The promoter sequences, promoter control elements or vectors of thepresent document may be transformed into host cells. Thesetransformations may be into protoplasts or intact tissues or isolatedcells. Preferably expression vectors are introduced into intact tissue.General methods of culturing plant tissues are provided for example byMaki et al. (1993) “Procedures for Introducing Foreign DNA into Plants”In Methods in Plant Molecular Biology & Biotechnology, Glich et al. Eds.pp. 67-88 CRC Press; and by Phillips et al. (1988) “Cell-Tissue Cultureand In-Vitro Manipulation” In Corn & Corn Improvement, 3rd EditionSprague et al. eds., pp. 345-387, American Society of Agronomy Inc. etal.

Methods of introducing polynucleotides into plant tissue include thedirect infection or co-cultivation of plant cell with Agrobacteriumtumefaciens, Horsch et al. (1985) Science, 227:1229. Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer provided by Gruber et al. supra.

Alternatively, polynucleotides are introduced into plant cells or otherplant tissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably polynucleotides are introduced into planttissues using the microprojectile media delivery with the biolisticdevice. See, for example, Tomes et al., “Direct DNA transfer into intactplant cells via microprojectile bombardment” In: Gamborg and Phillips(Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods,Springer Verlag, Berlin (1995).

Methods for specifically transforming dicots are well known to thoseskilled in the art. Transformation and plant regeneration using thesemethods have been described for a number of crops including, but notlimited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut(Arachis hypogaea), and members of the genus Brassica.

Methods for transforming monocots are well known to those skilled in theart. Transformation and plant regeneration using these methods have beendescribed for a number of crops including, but not limited to, barley(Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass(Dactylis glomerata); rice (Oryza sativa, including indica and japonicavarieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tallfescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostisstolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticumaestivum), switchgrass (Panicum vigatum) and alfalfa (Medicago sativa).It is apparent to those of skill in the art that a number oftransformation methodologies can be used and modified for production ofstable transgenic plants from any number of target plants of interest.

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp. or hybrids thereof, Sorghum spp.or hybrids thereof, sudangrass, Miscanthus spp. or hybrids thereof,Saccharum spp. or hybrids thereof, Erianthus spp., Populus spp.,Andropogon gerardii (big bluestem), Pennisetum purpureum (elephantgrass) or hybrids thereof (e.g., Pennisetum purpureum×Pennisetumtyphoidum), Phalaris arundinacea (reed canarygrass), Cynodon dactylon(bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata(prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giantreed) or hybrids thereof, Secale cereale (rye), Salix spp. (willow),Eucalyptus spp. (eucalyptus), Triticosecale (Triticum—wheat×rye),Tripsicum dactyloides (Eastern gammagrass), Leymus cinereus (basinwildrye), Leymus condensatus (giant wildrye), and bamboo.

In some embodiments, a suitable species can be a wild, weedy, orcultivated sorghum species such as, but not limited to, Sorghum almum,Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor(such as bicolor, guinea, caudatum, kafir, and durra), Sorghumbrachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum,Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande,Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghumlaxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum sudanensese, Sorghum timorense, Sorghumtrichocladum, Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, orhybrids such as Sorghum×almum, Sorghum×sudangrass or Sorghum×drummondii.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava).

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (=Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinusalbus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.),Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acerspp. (maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Loliumspp. (ryegrass) and Phleum pratense (timothy).

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a specific species (e.g., Saccharumsp.×Miscanthus sp., Panicum virgatum×Panicum amarum, Panicumvirgatum×Panicum amarulum, and Pennisetum purpureum×Pennisetumtyphoidum).

In another embodiment of the current document, expression constructs canbe used for gene expression in callus culture for the purpose ofexpressing marker genes encoding peptides or polypeptides that allowidentification of transformed plants. Here, a promoter that isoperatively linked to a polynucleotide to be transcribed is transformedinto plant cells and the transformed tissue is then placed oncallus-inducing media. If the transformation is conducted with leafdiscs, for example, callus will initiate along the cut edges. Oncecallus growth has initiated, callus cells can be transferred to callusshoot-inducing or callus root-inducing media. Gene expression will occurin the callus cells developing on the appropriate media: callusroot-inducing promoters will be activated on callus root-inducing media,etc. Examples of such peptides or polypeptides useful as transformationmarkers include, but are not limited to barstar, glyphosate,chloramphenicol acetyltransferase (CAT), kanamycin, spectinomycin,streptomycin or other antibiotic resistance enzymes, green fluorescentprotein (GFP), and β-glucuronidase (GUS), etc. Some of the promotersprovided in SEQ ID NOs: 1-18 will also be capable of sustainingexpression in some tissues or organs after the initiation or completionof regeneration. Examples of these tissues or organs are somaticembryos, cotyledon, hypocotyl, epicotyl, leaf, stems, roots, flowers andseed.

Integration into the host cell genome also can be accomplished bymethods known in the art, for example, by the homologous sequences orT-DNA discussed above or using the Cre-lox system (A. C. Vergunst et al.(1998) Plant Mol. Biol. 38:393).

6. USES OF THE PROMOTERS

6.1 Use of the Promoters to Study and Screen for Expression

The promoters of the present application can be used to furtherunderstand developmental mechanisms. For example, promoters that arespecifically induced during callus formation, somatic embryo formation,shoot formation or root formation can be used to explore the effects ofoverexpression, repression or ectopic expression of target genes, or forisolation of trans-acting factors.

The vectors of the present application can be used not only forexpression of coding regions but may also be used in exon-trap cloning,or promoter trap procedures to detect differential gene expression invarious tissues (see Lindsey et al. (1993) Transgenic Research 2:3347.Auch and Reth (1990) Nucleic Acids Research 18: 6743).

Entrapment vectors, first described for use in bacteria (Casadaban andCohen (1979) Proc. Nat. Aca. Sci. U.S.A. 76: 4530; Casadaban et al.(1980) J. Bacteriol. 143: 971) permit selection of insertional eventsthat lie within coding sequences. Entrapment vectors can be introducedinto pluripotent ES cells in culture and then passed into the germlinevia chimeras (Gossler et al. 1989) Science 244: 463; Skarnes (1990)Biotechnology 8: 827). Promoter or gene trap vectors often contain areporter gene, e.g., lacZ, lacking its own promoter and/or spliceacceptor sequence upstream. That is, promoter gene traps contain areporter gene with a splice site but no promoter. If the vector lands ina gene and is spliced into the gene product, then the reporter gene isexpressed.

Recently, the isolation of preferentially-induced genes has been madepossible with the use of sophisticated promoter traps (e.g. IVET) thatare based on conditional auxotrophy complementation or drug resistance.In one WET approach, various bacterial genome fragments are placed infront of a necessary metabolic gene coupled to a reporter gene. The DNAconstructs are inserted into a bacterial strain otherwise lacking themetabolic gene, and the resulting bacteria are used to infect the hostorganism. Only bacteria expressing the metabolic gene survive in thehost organism; consequently, inactive constructs can be eliminated byharvesting only bacteria that survive for some minimum period in thehost. At the same time, broadly active constructs can be eliminated byscreening only bacteria that do not express the reporter gene underlaboratory conditions. The bacteria selected by such a method containconstructs that are selectively induced only during infection of thehost. The IVET approach can be modified for use in plants to identifygenes induced in either the bacteria or the plant cells upon pathogeninfection or root colonization. For information on IVET see the articlesby Mahan et al. (1993) Science 259:686-688, Mahan et al. (1995) Proc.Natl. Acad. Sci. USA 92:669-673, Heithoff et al. (1997) Proc. Natl.Acad. Sci. USA 94:934-939, and Wang et al. (1996) Proc. Natl. Acad. Sci.USA 93:10434.

6.2 Use of the Promoters to Transcribe Genes of Interest

In one embodiment of the document, a nucleic acid molecule as shown inSEQ ID NOs: 1-12 is incorporated into a construct such that a promoterof the present document is operably linked to a transcribable nucleicacid molecule that is a gene of agronomic interest. As used herein, theterm “gene of agronomic interest” refers to a transcribable nucleic acidmolecule that includes but is not limited to a gene that provides adesirable characteristic associated with plant morphology, physiology,growth and development, yield, nutritional enhancement, disease or pestresistance, or environmental or chemical tolerance. The expression of agene of agronomic interest is desirable in order to confer anagronomically important trait. A gene of agronomic interest thatprovides a beneficial agronomic trait to crop plants may be, forexample, including, but not limited to genetic elements comprisingherbicide resistance, increased yield, increased biomass, insectcontrol, fungal disease resistance, virus resistance, nematoderesistance, bacterial disease resistance, starch production, modifiedoils production, high oil production, modified fatty acid content, highprotein production, fruit ripening, enhanced animal and human nutrition,biopolymers, environmental stress resistance, pharmaceutical peptides,improved processing traits, improved digestibility, industrial enzymeproduction, improved flavor, nitrogen fixation, hybrid seed production,and biofuel production. The genetic elements, methods, and transgenesdescribed in the patents listed above are hereby incorporated byreference.

Alternatively, a transcribable nucleic acid molecule can effect theabove mentioned phenotypes by encoding a RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense, inhibitory RNA (RNAi), or cosuppression-mediated mechanisms.The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)engineered to cleave a desired endogenous mRNA product. Thus, anynucleic acid molecule that encodes a protein or mRNA that expresses aphenotype or morphology change of interest may be useful for thepractice of the present document.

6.3. Stress Induced Preferential Transcription

Promoters and control elements providing modulation of transcriptionunder oxidative, drought, oxygen, wound, and methyl jasmonate stress areparticularly useful for producing host cells or organisms that are moreresistant to biotic and abiotic stresses. In a plant, for example,modulation of genes, transcripts, and/or polypeptides in response tooxidative stress can protect cells against damage caused by oxidativeagents, such as hydrogen peroxide and other free radicals.

Drought induction of genes, transcripts, and/or polypeptides are usefulto increase the viability of a plant, for example, when water is alimiting factor. In contrast, genes, transcripts, and/or polypeptidesinduced during oxygen stress can help the flood tolerance of a plant.

The promoters and control elements of the present document can modulatestresses similar to those described in, for example, stress conditionsare VuPLD1 (drought stress; Cowpea; see Pham-Thi et al. (1999) Plant MolBiol 39:1257-65), pyruvate decarboxylase (oxygen stress; rice; seeRivosal et al. (1997) Plant Physiol 114(3): 1021-29), chromoplastspecific carotenoid gene (oxidative stress; Capsicum; see Bouvier et al.(1998) J Biol Chem 273: 30651-59).

Promoters and control elements providing preferential transcriptionduring wounding or induced by methyl jasmonate can produce a defenseresponse in host cells or organisms. In a plant, for example,preferential modulation of genes, transcripts, and/or polypeptides undersuch conditions is useful to induce a defense response to mechanicalwounding, pest or pathogen attack or treatment with certain chemicals.

Promoters and control elements of the present document also can triggera response similar to those described for cf9 (viral pathogen; tomato;see O'Donnell et al. (1998) Plant J 14(1): 137-42), hepatocyte growthfactor activator inhibitor type 1 (HAI-1), which enhances tissueregeneration (tissue injury; human; Koono et al. (1999) J HistochemCytochem 47: 673-82), copper amine oxidase (CuAO), induced duringontogenesis and wound healing (wounding; chick-pea; Rea et al. (1998)FEBS Lett 437: 177-82), proteinase inhibitor II (wounding; potato; seePena-Cortes et al. (1988) Planta 174: 84-89), protease inhibitor II(methyl jasmonate; tomato; see Farmer and Ryan (1990) Proc Natl Acad SciUSA 87: 7713-7716), two vegetative storage protein genes VspA and VspB(wounding, jasmonic acid, and water deficit; soybean; see Mason andMullet (1990) Plant Cell 2: 569-579).

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease oxidative, flood, or drought tolerance may requireup-regulation of transcription.

Typically, promoter or control elements, which provide preferentialtranscription in wounding or under methyl jasmonate induction, producetranscript levels that are statistically significant as compared to celltypes, organs or tissues under other conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.4. Light Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by light exposure can be utilized to modulate growth,metabolism, and development; to increase drought tolerance; and decreasedamage from light stress for host cells or organisms. In a plant, forexample, modulation of genes, transcripts, and/or polypeptides inresponse to light is useful

-   -   (1) to increase the photosynthetic rate;    -   (2) to increase storage of certain molecules in leaves or green        parts only, e.g. silage with high protein or starch content;    -   (3) to modulate production of exogenous compositions in green        tissue, e.g. certain feed enzymes;    -   (4) to induce growth or development, such as fruit development        and maturity, during extended exposure to light;    -   (5) to modulate guard cells to control the size of stomata in        leaves to prevent water loss, or    -   (6) to induce accumulation of beta-carotene to help plants cope        with light induced stress.

The promoters and control elements of the present document also cantrigger responses similar to those described in: abscisic acidinsensitive3 (ABI3) (dark-grown Arabidopsis seedlings, see Rohde et al.(2000) Plant Cell 12: 35-52), asparagine synthetase (pea root nodules,see Tsai and Coruzzi (1990) EMBO J 9: 323-32), mdm2 gene (human tumor,see Saucedo et al. (1998) Cell Growth Differ 9: 119-30).

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease drought or light tolerance may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues or organs exposed to light, producetranscript levels that are statistically significant as compared tocells, tissues, or organs under decreased light exposure (intensity orlength of time).

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.5. Dark Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by dark or decreased light intensity or decreased light exposuretime can be utilized to time growth, metabolism, and development, tomodulate photosynthesis capabilities for host cells or organisms. In aplant, for example, modulation of genes, transcripts, and/orpolypeptides in response to dark is useful, for example,

-   -   (1) to induce growth or development, such as fruit development        and maturity, despite lack of light;    -   (2) to modulate genes, transcripts, and/or polypeptide active at        night or on cloudy days; or    -   (3) to preserve the plastid ultra structure present at the onset        of darkness.

The present promoters and control elements can also trigger responsesimilar to those described in the section above.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth and development may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription under exposure to dark or decrease light intensity ordecrease exposure time, produce transcript levels that are statisticallysignificant.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.6. Leaf Preferential Transcription

Promoters and control elements providing preferential transcription in aleaf can modulate growth, metabolism, and development or modulate energyand nutrient utilization in host cells or organisms. In a plant, forexample, preferential modulation of genes, transcripts, and/orpolypeptide in a leaf, is useful, for example,

-   -   (1) to modulate leaf size, shape, and development;    -   (2) to modulate the number of leaves; or    -   (3) to modulate energy or nutrient usage in relation to other        organs and tissues.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of a leaf, producetranscript levels that are statistically significant as compared toother cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.7. Root Preferential Transcription

Promoters and control elements providing preferential transcription in aroot can modulate growth, metabolism, development, nutrient uptake,nitrogen fixation, or modulate energy and nutrient utilization in hostcells or organisms. In a plant, for example, preferential modulation ofgenes, transcripts, and/or polypeptide in a root, is useful,

-   -   (1) to modulate root size, shape, and development;    -   (2) to modulate the number of roots, or root hairs;    -   (3) to modulate mineral, fertilizer, or water uptake;    -   (4) to modulate transport of nutrients; or    -   (4) to modulate energy or nutrient usage in relation to other        cells, organs and tissues.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs of a root, produce transcriptlevels that are statistically significant as compared to other cells,organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.8. Stem/Shoot Preferential Transcription

Promoters and control elements providing preferential transcription in astem or shoot can modulate growth, metabolism, and development ormodulate energy and nutrient utilization in host cells or organisms. Ina plant, for example, preferential modulation of genes, transcripts,and/or polypeptide in a stem or shoot, is useful, for example,

-   -   (1) to modulate stem/shoot size, shape, and development; or    -   (2) to modulate energy or nutrient usage in relation to other        organs and tissues

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of a stem or shoot,produce transcript levels that are statistically significant as comparedto other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.9. Fruit and Seed Preferential Transcription

Promoters and control elements providing preferential transcription in asilique or fruit can time growth, development, or maturity; or modulatefertility; or modulate energy and nutrient utilization in host cells ororganisms. In a plant, for example, preferential modulation of genes,transcripts, and/or polypeptides in a fruit, is useful

-   -   (1) to modulate fruit size, shape, development, and maturity;    -   (2) to modulate the number of fruit or seeds;    -   (3) to modulate seed shattering;    -   (4) to modulate components of seeds, such as, storage molecules,        starch, protein, oil, vitamins, anti-nutritional components,        such as phytic acid;    -   (5) to modulate seed and/or seedling vigor or viability;    -   (6) to incorporate exogenous compositions into a seed, such as        lysine rich proteins;    -   (7) to permit similar fruit maturity timing for early and late        blooming flowers; or    -   (8) to modulate energy or nutrient usage in relation to other        organs and tissues.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of siliques or fruits,produce transcript levels that are statistically significant as comparedto other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.10. Callus Preferential Transcription

Promoters and control elements providing preferential transcription in acallus can be useful to modulating transcription in dedifferentiatedhost cells. In a plant transformation, for example, preferentialmodulation of genes, transcripts, in callus is useful to modulatetranscription of a marker gene, which can facilitate selection of cellsthat are transformed with exogenous polynucleotides.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease marker gene detectability, for example, may requireup-regulation of transcription.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.11. Flower Specific Transcription

Promoters and control elements providing preferential transcription inflowers can modulate pigmentation; or modulate fertility in host cellsor organisms. In a plant, for example, preferential modulation of genes,transcripts, and/or polypeptides in a flower, is useful,

-   -   (1) to modulate petal color; or    -   (2) to modulate the fertility of pistil and/or stamen.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease pigmentation, for example, may requireup-regulation of transcription

Typically, promoter or control elements, which provide preferentialtranscription in flowers, produce transcript levels that arestatistically significant as compared to other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.12. Immature Bud/Floret and Inflorescence Preferential Transcription

Promoters and control elements providing preferential transcription inan immature bud/floret or inflorescence can time growth, development, ormaturity; or modulate fertility or viability in host cells or organisms.In a plant, for example, preferential modulation of genes, transcripts,and/or polypeptide in an immature bud and/or inflorescence, is useful,

-   -   (1) to modulate embryo development, size, and maturity;    -   (2) to modulate endosperm development, size, and composition;    -   (3) to modulate the number of seeds and fruits; or    -   (4) to modulate seed development and viability.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in immature buds/florets and inflorescences, producetranscript levels that are statistically significant as compared toother cell types, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.13. Senescence Preferential Transcription

Promoters and control elements providing preferential transcriptionduring senescence can be used to modulate cell degeneration, nutrientmobilization, and scavenging of free radicals in host cells ororganisms. Other types of responses that can be modulated include, forexample, senescence associated genes (SAG) that encode enzymes thoughtto be involved in cell degeneration and nutrient mobilization(Arabidopsis; see Hensel et al. (1993) Plant Cell 5: 553-64), and theCP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol Reprod 57:1467-77), both induced during senescence.

In a plant, for example, preferential modulation of genes, transcripts,and/or polypeptides during senescencing is useful to modulate fruitripening.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease scavenging of free radicals, for example, mayrequire up-regulation of transcription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs during senescence, producetranscript levels that are statistically significant as compared toother conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

6.14. Germination Preferential Transcription

Promoters and control elements providing preferential transcription in agerminating seed can time growth, development, or maturity; or modulateviability in host cells or organisms. In a plant, for example,preferential modulation of genes, transcripts, and/or polypeptide in agerminating seed, is useful,

-   -   (1) to modulate the emergence of the hypocotyls, cotyledons and        radical; or    -   (2) to modulate shoot and primary root growth and development;

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in a germinating seed, produce transcript levels that arestatistically significant as compared to other cell types, organs ortissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7. EXPERIMENTAL PROCEDURES AND RESULTS

Agrobacterium-Mediated Transformation of Rice

Induce Calli Formation from Mature Rice Seeds

De-husk mature seeds using de-husker (Kett; Cat #TR120) and discardspotted ones if present. Transfer 100 de-husked seeds to a 50 mL conicaltube. Add 24 mL autoclaved distilled water and then 6 ml CLOROX™(CLOROX™ contains 5.25% sodium hypochlorite so final concentration is1.05%) and 2-3 drops of LIQUI-NOX®. Shake the tube occasionally for 30min. Pour out CLOROX™ solution and rinse seeds 5 times with sterilewater. Dry the seeds on autoclaved KIMWIPES™ for a few minutes. Transferseeds on the semi-solid N6-P medium (3.98 g/L N6 basal salt mixture, 0.8mg/L KI, 0.025 mg/L CoCl2.6H20, 0.025 mg/L CuSO4.5H20, 0.25 mg/LNaMoO4.2H20, 2 mg/L Glycine, 100 mg/L Myo-inositol, 5 mg/L Thiamine.HCl,1 mg/L Pyridoxine.HCl, 1 mg/L Nicotinic acid, 2.8 g/L Proline, 300 mg/LCasamino acid, 30 g/L Sucrose, 2 mg/L 2, 4-Dichloro-Phenoxyacetic Acid,4 g/L Gel rite, pH 5.6); 10 seeds are placed in each Petri dish. EachPetri dish contains 30 ml N6-P medium. Dishes are sealed with antifungaltape to allow air exchange. Place the plates at 28° C. under coldfluorescent light. Many granular calli should be formed within 4 weeks.Calli of good quality consist of small and spherical cells with densecytoplasm, which are competent for transformation. The calli can be useddirectly for Agrobacterium infection, or subculture them for later use.

Infection and Co-Cultivation of Calli with Agrobacterium Cells

Pick up a single Agrobacterium clone from stock and culture it in 2 mLYEB liquid medium by growing it overnight in a shaker. Appropriateantibiotics are included at 50 mg/L or higher. Put on 28° C. shakerovernight. The next day, reinoculate 25 μL of overnight culture into 5mL of liquid YEB with selection and grow overnight at 28° C. The nextday, use this culture for transformation. Transfer the liquid culture to1.5 mL microtube and centrifuge it at 10,000 RPM for 2 minutes. Discardthe supernatant and resuspend cells in 15 mL N6-AS liquid medium (3.98g/L N6 basal salt mixture, 0.8 mg/L KI, 0.025 mg/L CoCl₂.6H₂0, 0.025mg/L CuSO₄.5H₂0, 0.25 mg/L NaMoO₄.2H₂0, 2 mg/L Glycine, 100 mg/LMyo-inositol, 5 mg/L Thiamine.HCl, 1 mg/L Pyridoxine.HCl, 1 mg/LNicotinic acid, 1 g/L Casamino acid, 30 g/L Sucrose, 10 g/L Glucose, 2mg/L 2,4-Dichloro-Phenoxyacetic Acid, pH 5.6). Adjust cell density withthe N6-AS medium. The density can be measured by a spectrophotometer(use 750 μL aliquot for reading). The optimal OD₆₀₀ reading variessignificantly depending on Agrobacterium strains and sometimes vectors.The optimal reading means that there is no over-growth of Agrobacteriumcells in at least 3 day co-cultivation. The optimum OD₆₀₀ for rice is0.2. Mix rice calli with Agrobacterium cells. This is done in a 50 mLsterile conical tube. Place tubes in a sterile 1 gallon plastic bag andplace horizontally on a shaker for 30 minutes. Pipette out solution with10-mL pipette. Transfer calli onto autoclaved Kimwipes™ to remove excesssolution. Remove any agar media if present, since Agrobacterium willgrow faster in agar. Culture calli on autoclaved filter paper placed onthe N6-AS semi-solid medium (N6-AS medium containing 4 g/L Gel rite).Each 100 mm×20 mm dish contains 10-20 mL N6-AS media. Seal the dish withantifungal tape. Cover plates with aluminum foil because acetosyringonepresent in the media is light sensitive. Co-culture calli andAgrobacterium cells at 22° C. in the dark until Agrobacterium mass canbe seen by the naked eye.

Selection of Transformed Calli

Transfer the infected calli with a sterile, disposable spatula to asterile 50 mL tube containing 35 mL sterile distilled water. Shake thetube for a few seconds and pipette out the water. Repeat washing 3 timesor more if necessary, until solution becomes clear. For the final wash,add carbenicillin at the concentration of 500 mg/L. Transfer calli witha disposable blue ino-loop onto 2 layers thick of autoclaved KIMWIPES™paper in a large spherical Petri plate to blot. Culture calli on dishescontaining semisolid N6-P medium with 250 mg/L carbenicillin at 28° C.under cold fluorescent light for 6 days. Each 100 mm×20 mm dish contains30 mL N6-P medium; use approximately 4 dishes of calli for eachconstruct. Dishes are sealed with antifungal tape. Transfer calli fromthe resting media on to the selection media containing 250 mg/Lcarbenicillin and 5 mg/L purified BIALAPHOS™ (for selection of BAR gene)or 100 mg/L Paromomycin sulfate (for selection of NPTII gene) for 14days. For the second round of selection, subculture calli for another 14days. Transformed calli can typically be seen clearly at the end of thisselection. Third selection is done if the second selection does notproduce enough resistant calli.

Regeneration of Transgenic Plants

Transfer independent resistant calli to the N6-R plant regenerationmedium (3.98 g/L N6 basal salt mixture, 0.8 mg/L KI, 0.025 mg/LCoCL₂.6H₂0, 0.025 mg/L CuSO₄.5H₂0, 0.25 mg/L NaMoO₄.2H₂0, 2 mg/LGlycine, 100 mg/L Myo-inositol, 5 mg/L Thiamine.HCl, 1 mg/LPyridoxine.HCl, 1 mg/L Nicotinic acid, 1 g/L Casamino acid, 25 g/LSucrose, 25 g/L Sorbitol, 2 mg/L 6-Benzylaminopurine, 0.05 mg/L1-Naphthaleneacetic acid, 7 g/L Agarose (Omnipur), pH 5.6). Each 100mm×20 mnm dish contains 30 mL N6-R medium, 4 or 6 callus lines on eachdish. Dishes are sealed with antifungal tape. Culture calli at 28° C.under cold fluorescent light until shoots and roots are formed.Typically, shoots should be seen within 3 weeks. Transfer plantlets toMagenta boxes containing 30 mL ½ MS1A (2.165 g/L MS salts, 1 ml/L of1000×B5 vitamins stock, 15 g/L Sucrose, 5 g/L Agar, pH 5.7). Growplantlets at 28° C. under old fluorescent light for 10-14 days.

Ten independently transformed events (plantlets) are selected with onetiller from each event evaluated for GFP Expression in the T0generation.

Preparation of Soil Mixture: 6 L Potting Soil (Farmers Organic PottingSoil, Chino, Calif.) is mixed with 4 L Turface in a cement mixer to makea 60:40 soil mixture. To the soil mixture is added 1 tsp Marathon 1%granules (Hummert, Earth City, Mo.), 2 Tbsp OSMOCOTE® 14-14-14 (Hummert,Earth City, Mo.). Once a month 1 Tbsp Peters fertilizer 20-20-20 (J.R.Peters, Inc., Allentown, Pa.) is mixed well in 3 gallons of water andpoured into the bottom flat to fertilize. 6-inch diameter Azalea potsare used for transplanting with 1-2 plants per pot.

Planting: Plants growing in magenta boxes are carefully pulled from MSagar rice media. Plant roots are cleaned and divided into single tillersensuring that each tiller has a viable root and no residual callusmaterial. The tillers are screened for GFP expression and one positivelyexpressing tiller per independently transformed event is transplanted tosoil and grown to maturity for further analysis.

Plant Maintenance: Plants are well watered throughout the duration ofthe lifecycle. The bottom of the flat is cleaned and new water addedtwice a week. Approximately 21 days after planting, rice issub-irrigated with Peter's fertilizer at a concentration of 1 Tsp per 3gallons of water. Plants are analyzed for GFP expression at the T0seedling, T0 mature and T1 generations.

GFP Assay and Imaging

The polynucleotide sequences of the present document were tested forpromoter activity using Green Fluorescent Protein (GFP) assays in thefollowing manner.

Each isolated nucleic acid described in the Sequence Listing was clonedinto a Ti plasmid vector, CRS380_Binary_DF_EGFP using appropriateprimers tailed with SfiI restriction sites. Standard PCR reactions usingthese primers and genomic DNA were conducted. The resulting product wasisolated, cleaved with SfiI and cloned into the SfiI site of anappropriate vector, such as, CRS380_Binary_DF_EGFP (see FIG. 1).

GFP Assay in Rice Callus

GFP expression in rice callus can be observed as early as 4-7 days afterco-cultivation. The rice callus used for co-cultivation is observedunder Zeiss Stemi SVII APO dissecting microscope for GFP expression. Forviewing GFP expression we use GFP 500 filter in the microscope. Theimages observed under the microscope can be transferred, captured andstored to a computer using the Axiocam (Zeiss) camera and Axiovisionsoftware.

GFP Assay in T0 Seedling

Each independently transformed event is divided into single tillerswhich then undergo Typhoon scanner laser imaging. One positivelyGFP-expressing tiller per event is selected for subsequent GFP analysisby Confocal microscopy and ultimately for transplantation for furthermature tissue analysis.

Typhoon Scan: Plants are initially scanned with a Typhoon Scanner toexamine the GFP expression of the plants on a global level. Ifexpression is present, images are collected by Typhoon scanning laserimaging and scanning laser confocal microscopy. Scanned images from theTyphoon scanner are taken as 2-D images of the entire plant and can beopened using the program ImageQuant.

Confocal Microscopy: Tissues are dissected by eye or under magnificationusing INOX 5 grade forceps and placed on a slide with water andcoversliped. An attempt is made to record images of observed expressionpatterns at earliest and latest stages of development of tissues listedbelow. Specific tissues will be defined as having positive expression orno expression.

Main Culm Bundle sheath, endodermis, epidermis, internode, ligule, node,not-specific, pericycle, phloem, sclerenchyma layer, vasculature, xylem.Root Cortex, epidermis, not-specific, root cap, vascular. Panicle Flagleaf, not-specific, ovary, peduncle, primary branch, rachilla, rachis,spikelet Spikelet Aleurone layer, anther, carpel, embryo, endosperm,filament, flag leaf, floret (palea), leamma, not-specific, ovule,pedicle, pollen, seed, stigma Leaf Epidermis, leaf blade, leaf sheath,magin, mesophyll, not-specific, petiole, primordia, stipule, stomata,trichome, vasculature Meristem Floral meristem, not-specific, shootapical meristem, vegetative meristem

Ziess UV stereoscope: Reproductive tissues that are too large to usewith the confocal microscope are prepared using a dissection microscopeunder high magnification using INOX 5 grade forceps and placed on aslide. An attempt is made to record images of observed expressionpatterns in mature rice reproductive tissues. Auxiovision is the programused for capturing GFP images under the following settings:

GFP: 4900 ms, gain=3, resolution=1300×1030 interpolated

Bright field: 100 ms, conversion=square root, resolution=1300×1030interpolated.

T0 Mature

These are the T0 plants resulting from a single tiller from eachindependent transformation event having predetermined positive GFPexpression. These are screened between stage 3-5 (i.e. between latevegetative to panicle initiation and floral maturation), which is 6-8weeks of age. At this stage the mature plant possesses young panicleinflorescence to adolescent flowers, fully expanded leaves, multiplenodes and mature stem and root tissue. The plants are initially imagedusing the Typhoon scanner and then imaged in detail using the LeicaConfocal microscope and Ziess UV Stereoscope to allow examination of themature plants on a global level.

T1 Seedling

Seed is collected from the T0 plants and stored for further use ininduction experiments.

Results

The Promoter Expression Reports of the Tables present the results of theGFP assays as reported by their corresponding construct number and linenumber.

Derivatives of PD3525, PD3559, PD3560, PD3561, PD3562, PD3564, PD3565,PD3573, PD3574, PD3578, PD3579, PD3580, PD3567, PD3655, PD3720, PD3786,PD3805, or PD3812 are generated by introducing mutations into thenucleotide sequence set forth in SEQ ID NO:1-SEQ ID NO:18 as disclosedin U.S. Pat. No. 6,747,189, incorporated herein by reference. Aplurality of mutagenized DNA segments derived from PD3525, PD3559,PD3560, PD3561, PD3562, PD3564, PD3565, PD3573, PD3574, PD3578, PD3579,PD3580, PD3567, PD3655, PD3720, PD3786, PD3805, or PD3812, includingderivatives with nucleotides deletions and modifications are generatedand inserted into a plant transformation vector operably linked to a GFPmarker gene. Each of the plant transformation vectors are preparedessentially as described above, except that the full length promoter isreplaced by a mutagenized derivative. Plants (e.g., rice plants) aretransformed with each of the plant transformation vectors and analyzedfor expression of the GFP marker to identify those mutagenizedderivatives having promoter activity.

Fragments of PD3525, PD3559, PD3560, PD3561, PD3562, PD3564, PD3565,PD3573, PD3574, PD3578, PD3579, PD3580, PD3567, PD3655, PD3720, PD3786,PD3805, or PD3812 are isolated by designing primers to clone fragmentsof the promoters set forth in SEQ ID NO:1-18. A plurality of clonedfragments of PD3525, PD3559, PD3560, PD3561, PD3562, PD3564, PD3565,PD3573, PD3574, PD3578, PD3579, PD3580, PD3567, PD3655, PD3720, PD3786,PD3805, or PD3812 ranging in size from 50 nucleotide up to the fulllength sequence set forth in SEQ ID NO:1-18 are obtained using PCR. Forexample, a fragment of PD3525 of about 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, or 990nucleotides in length from various parts of PD3525 (SEQ ID NO:1) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3535 can include one or moreof a GARE1OSREP1, ACIIPVPAL2, ABRERATCAL, and TATABOX motif.

A fragment of PD3559 of about 50, 75, 100, 125, 150, 175, 190, or 200nucleotides in length from various parts of PD3559 (SEQ ID NO:2) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3559 can include one or moreof an AUXRETGA2GMGH3, ACGTABREMOTIFA2OSEM, TATCCAYMOTIFOSRAMY3D,AMYBOX1, and GAREAT motif.

A fragment of PD3560 of about 50, 100, 150, 200, 250, 275, 300, 350,375, or 390 nucleotides in length from various parts of PD3560 (SEQ IDNO:3) are obtained and inserted into a plant transformation vectoroperably linked to a GFP marker gene. Such fragments of PD3560 caninclude one or more of an ARE1, SBOXATRBCS, TE2F2NTPCNA, GADOWNAT,ACGTABREMOTIFA2OSEM, and TATABOX motif.

A fragment of PD3561 of about 50, 100, 150, 200, 250, 275, 300, 350,375, 390, 400, or 410 nucleotides in length from various parts of PD3561(SEQ ID NO:4) are obtained and inserted into a plant transformationvector operably linked to a GFP marker gene. Such fragments of PD3561can include one or more of a ROOTMOTIFTAPOX1, RYREPEATVFLEB4, andTATABOX motif.

A fragment of PD3562 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 725, or 750 nucleotides in length fromvarious parts of PD3562 (SEQ ID NO:5) are obtained and inserted into aplant transformation vector operably linked to a GFP marker gene. Suchfragments of PD3562 can include one or more of a CARGCW8GAT, INRNTPSADB,and TATABOX2 motif.

A fragment of PD3564 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,or 1010 nucleotides in length from various parts of PD3564 (SEQ ID NO:6)are obtained and inserted into a plant transformation vector operablylinked to a GFP marker gene. Such fragments of PD3564 can include one ormore of a MARTBOX, RYREPEATVFLEB4, NRRBNEXTA, TRANSINITMONOCOTS,TATABOXOSPAL, and P1BS motif.

A fragment of PD3565 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1010, 1025, 1050, or 1060 nucleotides in length from various parts ofPD3565 (SEQ ID NO:7) are obtained and inserted into a planttransformation vector operably linked to a GFP marker gene. Suchfragments of PD3565 can include one or more of a CARGCW8GAT, P1BS, andTATABOX motif.

A fragment of PD3573 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, or 1000nucleotides in length from various parts of PD3573 (SEQ ID NO:8) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3573 can include one or moreof a ROOTMOTIFTAPOX1, ATHB6COREAT, TATABOX2, and UP2ATMSD motif.

A fragment of PD3574 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, or 1400 nucleotides in lengthfrom various parts of PD3574 (SEQ ID NO:9) are obtained and insertedinto a plant transformation vector operably linked to a GFP marker gene.Such fragments of PD3574 can include one or more of a PRECONSCRHSP70A,IBOXCORENT, AGCBOXNPGLB, UP2ATMSD, and TATABOX4 motif.

A fragment of PD3578 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200,2250, 2300, 2350, 2400, 2450, or 2475 nucleotides in length from variousparts of PD3578 (SEQ ID NO:10) are obtained and inserted into a planttransformation vector operably linked to a GFP marker gene. Suchfragments of PD3578 can include one or more of an IBOXCORENT, ERELEE4,ABREATRD22, P1BS, and TATABOX motif.

A fragment of PD3579 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, or 2075nucleotides in length from various parts of PD3579 (SEQ ID NO:11) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3579 can include one or moreof a CACGCAATGMGH3 and UPRMOTIFIIAT motif. For example, a fragment ofPD3579 can contain nucleotides 1 to 896 of SEQ ID NO:11.

A fragment of PD3580 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900, 1950, or 1975 nucleotides in lengthfrom various parts of PD3580 (SEQ ID NO:12) are obtained and insertedinto a plant transformation vector operably linked to a GFP marker gene.Such fragments of PD3580 can include one or more of a PRECONSCRHSP70A,ACIIPVPAL2, TATABOX4, CAAT-box, CAATBOX1, and UPRMOTIFIIAT motif. Forexample, a fragment can contain nucleotides 501 to 2000 of SEQ ID NO:12.

A fragment of PD3567 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, or 1275 nucleotides in length from variousparts of PD3567 (SEQ ID NO:13) are obtained and inserted into a planttransformation vector operably linked to a GFP marker gene. Suchfragments of PD3567 can include one or more of a RYREPEATVFLEB4,SPHCOREZMC1, and AGCBOXNPGLB motif.

A fragment of PD3655 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 975nucleotides in length from various parts of PD3655 (SEQ ID NO:14) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3655 can include one or moreof a ROOTMOTIFTAPOX1, RYREPEATVFLEB4, P1BS, and TATABOX1 motif. Forexample, a fragment of PD3655 can contain nucleotides 501 to 990 of SEQID NO:14.

A fragment of PD3720 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1475nucleotides in length from various parts of PD3720 (SEQ ID NO:15) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3720 can include one or moreof a SBOXATRBCS, MYBbindingsite, ATHB1ATCONSENSUS, ABRE, E2FAT,PRECONSCRHSP70A, MYBPLANT, and E2FCONSENSUS motif. For example, afragment of PD3720 can contain nucleotides 702 to 1500 of SEQ ID NO:15.

A fragment of PD3786 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, or 1875 nucleotides in length from variousparts of PD3786 (SEQ ID NO:16) are obtained and inserted into a planttransformation vector operably linked to a GFP marker gene. Suchfragments of PD3786 can include one or more of a P1BS, ABREZMRAB28,SP8BFIBSP8BIB, CCA1ATLHCB1, BOXIIPCCHS, and LRENPCABE motif.

A fragment of PD3805 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, or 990nucleotides in length from various parts of PD3805 (SEQ ID NO:17) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3805 can include one or moreof a P1BS, MYBGAHV, and CEREGLUBOX2PSLEGA motif.

A fragment of PD3812 of about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, 990, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1475nucleotides in length from various parts of PD3812 (SEQ ID NO:18) areobtained and inserted into a plant transformation vector operably linkedto a GFP marker gene. Such fragments of PD3812 can include one or moreof a CACGCAATGMGH3 or UPRMOTIFIIAT motif. For example, a fragment ofPD3812 can contain nucleotides 353 to 1248 of SEQ ID NO:18.

Each of the plant transformation vectors are prepared essentially asdescribed above except that the full length sequence is replaced by afragment containing one or more of the motifs described herein.Arabidopsis plants are transformed with each of the plant transformationvectors and analyzed for expression of the GFP marker to identify thosefragments having promoter activity.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the document can be made. Such modificationsare to be considered within the scope of the document as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

What is claimed is:
 1. A nucleic acid comprising a first nucleic acidsequence operably linked to a second nucleic acid sequence heterologousto said first nucleic acid sequence, said first nucleic acid sequencehaving 95 percent or greater sequence identity to the nucleotidesequence set forth in SEQ ID NO:12 or a fragment of SEQ ID NO:12 atleast 1450 nucleotides in length, said first nucleic acid sequencecomprising a PRECONSCRHSP70A motif, an ACIIPVPAL2 motif, a TATABOX4motif, and a CAAT-BOX motif, wherein said first nucleic acid sequence orsaid fragment directs transcription of said second nucleic acidsequence.
 2. The nucleic acid of claim 1, wherein said sequence identityis 98 percent or greater.
 3. The nucleic acid of claim 1, wherein saidnucleic acid sequence further contains a CAATBOX1 motif and anUPRMOTIFIIAT motif.
 4. The nucleic acid according to claim 1, whereinsaid first nucleic acid sequence consists of the nucleotide sequence setforth in SEQ ID NO: 12 or nucleotides 501-2000 of SEQ ID NO:12.
 5. Thenucleic acid according to claim 1, wherein said second nucleic acidsequence encodes a polypeptide.
 6. The nucleic acid according to claim5, wherein said second nucleic acid sequence is operably linked to saidfirst nucleic acid sequence in sense orientation.
 7. The nucleic acidaccording to claim 1, wherein said second nucleic acid sequence isoperably linked to said first nucleic acid sequence in antisenseorientation.
 8. The nucleic acid according to claim 7, wherein saidsecond nucleic acid sequence is transcribed into an antisense RNAmolecule.
 9. The nucleic acid according to claim 1, wherein said secondnucleic acid sequence is transcribed into an interfering RNA against anendogenous gene.
 10. A plant or plant cell transformed with the nucleicacid according to claim
 1. 11. The plant or plant cell of claim 10,wherein said first nucleic acid sequence consists of the nucleotidesequence set forth in SEQ ID NO: 12 or nucleotides 501-2000 of SEQ IDNO:12.
 12. A method of directing transcription in a plant cellcomprising transforming a plant cell with said nucleic acid according toclaim
 1. 13. The method of claim 12, wherein said first nucleic acidsequence consists of the nucleotide sequence according to SEQ ID NO:12.14. The method of claim 12, wherein said transformed plant cell isregenerated into a transformed plant comprising said first and secondnucleic acids.
 15. A transgenic plant comprising the nucleic acidaccording to claim
 1. 16. A transgenic plant according to claim 15,wherein said second nucleic acid encodes a polypeptide of agronomicinterest.
 17. A seed of the plant according to claim 15, said seedcomprising said nucleic acid.
 18. A method of expressing an exogenouscoding region in a plant comprising: (a) transforming a plant cell withthe nucleic acid of claim 6; (b) regenerating a stably transformed plantfrom the transformed plant cell of step (a); and (c) selecting plantscontaining said transformed plant cell of step (a), wherein expressionof the nucleic acid results in production of a polypeptide encoded bysaid second nucleic acid.
 19. A plant obtained according to the methodof claim
 18. 20. A transgenic seed obtained from the plant according toclaim 19, wherein said seed comprises said nucleic acid.
 21. A method ofproducing a transgenic plant, said method comprising: (a) introducinginto a plant cell said nucleic acid according to claim 1, and (b)growing a plant from said plant cell, wherein said plant comprises saidnucleic acid.
 22. The method of claim 21, wherein said second nucleicacid sequence encodes a polypeptide.
 23. The method of claim 21, whereinsaid second nucleic acid sequence is operably linked to said firstnucleic acid sequence in the antisense orientation.
 24. The method ofclaim 21, wherein said second nucleic acid sequence is transcribed intoan interfering RNA.